JP7635208B2 - Hepatitis B vaccine - Google Patents

Description

STATEMENT OF GOVERNMENT INTEREST Pursuant to Contract No. 89233218CNA000001 between the United States Department of Energy and TRIAD National Security, LLC for the operation of Los Alamos National Laboratory, the United States Government has certain rights in this invention.

SEQUENCE LISTING STATEMENT The sequence listing accompanying this application is provided in text format as an alternative to the written form and is hereby incorporated by reference. The name of the text file containing the sequence listing is 930185_414WO_SEQUENCE_LISTING.txt. The text file is 186 KB, was created on August 21, 2020, and has been submitted electronically via EFS-Web.

FIELD OF THE PRESENT FIELD OF THE PRESENT SUBJECT MATTER The present subject matter relates generally to Hepatitis B Virus (HBV), and specifically to HBV vaccines.

2. Description of Related Art
Hepatitis B virus (HBV) infection is a major health problem worldwide. HBV infection causes chronic liver disease in 5-10% of infected adults, whereas perinatal transmission reverses the trend, progressing to chronic disease in >90%. If untreated, chronic hepatitis B (CHB) infection often progresses to necrotizing inflammation and ongoing liver damage leading to cirrhosis and hepatocellular carcinoma (HCC). CHB is estimated to increase the risk of developing HCC 20-fold and accounts for approximately 54% of HCC cases. HCC is the third most common and deadly form of cancer, with approximately 800,000 new cases diagnosed each year. Highly effective (>95%) prophylactic vaccines have been available since the early 1980s; however, these HBV vaccines are less effective once infection has been established. Despite the widespread use of a preventive vaccine, there are 240-340 million chronic HBV carriers worldwide and more than 780,000 associated deaths per year. The majority of new HBV infections occur in highly endemic areas, such as China, Southeast Asia, and sub-Saharan Africa. HBV infection is transmitted sexually, in hospitals, or through blood.

The HBV genome is a 3.2 kB double-stranded DNA molecule that is composed of four overlapping open reading frames: polymerase (P or Pol), core (C), surface antigen (HBsAg) (S), and a gene called "X" whose function is not fully understood but which is involved in the development of liver cancer. Locarnini et al. Antivir Ther. 2010;15 Suppl 3:3-14. Also, unlike other small chronic viruses, such as HIV or HCV, which show a high mutation rate leading to quasispecies, the small (3.2 kb) DNA genome of HBV is more suppressed in its ability to mutate due to overlapping open reading frames (ORFs). Although mutation within the host is limited, different genotypes of HBV are found worldwide, which must be taken into account when designing treatments, e.g. vaccines.

The current standard of care for chronic HBV infection is treatment with antiviral drugs and interferon-alpha.

To date, therapeutic vaccination against HBV has not been effective. There are several potential reasons for this. Identifying the HBV sequence infecting an individual is useful in determining which of the specific vaccines will provide effective treatment. The HBV database, called "HBVdb," developed as a collaborative consortium to sequence and study the HBV genome, has identified approximately 5,000 complete genomes of HBV. See Hayer et al. Nucleic Acids Res., 41:D566-D570 2013, which is incorporated herein by reference in its entirety. However, due to overall overlap of sequences and isolation of multiple sequences from the same patient or from highly tight clusters, the total number of truly unique HBV complete genomes identified by HBVdb is approximately 3,000. The HBV genomes identified by HBVdb can be classified into different genotypes (A, B, C, CB, D, DC, DE, E, and F). The distribution of genotypes varies among people around the world. For example, approximately 75% of the dataset sequences available in the HBVdb in samples from individuals of Asian origin, and especially from China, contain genotypes B and C. On the other hand, more than half of HBV infections in Europe are caused by genotypes A and D.

Treatment with antiviral drugs and interferon alpha inhibits viral replication and stimulates the innate immune system, but they rarely eliminate the virus (1-4% per year) and patients often require lifelong treatment. Furthermore, individual responses to these therapies vary as the disease progresses, and prolonged treatment can lead to both resistance mutations and extensive side effects. Tenofovir and entecavir are the most effective direct antiviral therapies, but they are expensive and are used only in a subset of patients using expert guidelines and algorithms. If treatment fails or is not used, liver inflammation becomes chronic and a cycle of injury and regeneration can lead to fibrosis, abnormal liver structure, and in some cases HCC. Thus, there is an urgent need for effective immunotherapy that induces an effective immune response leading to elimination and eventual cure of HBV.

A small percentage of individuals with chronic HBV spontaneously clear the infection, as evidenced by no measurable surface antigen in the blood. The rate is as low as 1% per year, and improvement is minimal with direct-acting antiviral agents. The most effective clearance occurs in individuals with low quantitative levels of circulating surface antigen who are treated with type I interferon, which has severe side effects and is often poorly tolerated.

Therapeutic vaccinations are also not completely effective because they fail to lead to effective clearance of HBV in the liver, as the T cells required to clear the infection are exhausted or tolerized. Therefore, it is necessary to induce T cells that recognize peptide sequences not induced by natural infection but that are nevertheless displayed on the surface of infected hepatocytes.

A CMV/HBV vaccine provides a means to elicit an effective response leading to HBV elimination and eventual cure. Induction of active liver-resident effector CD8+ T cell (TEM) responses is critical for HBV elimination. Thus, induction of persistent HBV-specific effector CD8+ T cells recognizing novel epitopes in the liver of CHB patients should be sufficient to control and ultimately eliminate HBV.

Thus, there remains a need in the art to identify effective vaccinations for people infected with various genotypes of HBV. Also, because currently available vaccines are not effective against chronic HBV infection, there remains a need in the art to develop vaccines that can be used to treat such infections. There also remains a need to design, manufacture, and test therapeutic HBV vaccines in preparation for clinical trials. The compositions and methods disclosed herein address these needs.

Locarnini et al. Antivir Ther. 2010;15 Suppl 3:3-14 Hayer et al. Nucleic Acids Res., 41:D566-D570 2013

SUMMARY In certain aspects, the disclosure provides a polypeptide comprising an amino acid sequence as set forth in SEQ ID NOs:1-11 or SEQ ID NOs:14-36. In some embodiments, the polypeptide comprises two or more of the amino acid sequences as set forth in SEQ ID NOs:16-36. In some embodiments, the aforementioned polypeptides, or polynucleotides encoding the polypeptides, may be used in an HBV vaccine. In some embodiments, the disclosure provides an immunogenic composition comprising a polypeptide comprising an amino acid sequence as set forth in SEQ ID NOs:1-11 or SEQ ID NOs:14-36. In still further embodiments, the disclosure provides the use of the aforementioned polypeptides, or immunogenic compositions comprising the polypeptides, to elicit an immune response against HBV or to treat or prevent HBV infection.

In some embodiments, the disclosure provides viral vectors, including cytomegalovirus (CMV) vectors, comprising a polynucleotide comprising a sequence encoding one or more of the amino acid sequences set forth in SEQ ID NOs: 1-36. In some embodiments, the polynucleotide encodes one or more of the amino acid sequences comprising one or more of SEQ ID NOs: 1-11, SEQ ID NOs: 14-15, and SEQ ID NOs: 24-26. In some embodiments, the sequence encoded by the polynucleotide is sequenced to enhance expression. In some embodiments, the disclosure provides an immunogenic composition comprising a CMV vector encoding a polynucleotide comprising a sequence encoding one or more of the amino acid sequences set forth in SEQ ID NOs: 1-36. In yet further embodiments, the disclosure provides the use of the aforementioned vectors, or immunogenic compositions comprising the vectors, to generate an immune response against HBV or to treat or prevent HBV infection.

Also provided herein are immunogenic compositions or vaccines comprising one or more HBV episensus antigens. In some embodiments, the antigen of the vaccine is provided as a polypeptide. In some embodiments, the antigen is encoded by a polynucleotide. In some embodiments, a viral vector comprises the polynucleotide. In some embodiments, the antigen is encoded by two or more polynucleotides, where the polynucleotides may be expressed by the same promoter or separate promoters. In some embodiments, the antigens are encoded by separate viral vectors. In some embodiments, the HBV vaccine comprises two or more HBV episensus antigens. In some embodiments, the HBV vaccine comprises a cytomegalovirus (CMV) vector and a polynucleotide encoding one or more HBV episensus antigens.

In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine.

In some embodiments, the immunogenic composition or vaccine further comprises a pharma- ceutically acceptable carrier or excipient.

In one aspect, the present disclosure provides a composition, e.g., an immunogenic composition, that includes two or more of the aforementioned polypeptides, polynucleotides, vectors, or vaccines.

Also provided herein is a method of treating HBV in a subject, comprising administering to a subject in need thereof an effective amount of the aforementioned polypeptide, vector, vaccine, or composition. Further provided herein is a method of protecting a subject from HBV infection, comprising administering to a subject in need thereof an effective amount of the aforementioned polypeptide, vector, vaccine, or composition. Further provided herein is a method of eliciting an immune response against HBV, comprising administering to a subject in need thereof an effective amount of the aforementioned polypeptide, vector, vaccine, or composition.

Also provided herein are some embodiments in which the aforementioned polypeptide, vector, vaccine, or composition is for use in treating HBV, protecting a subject from HBV infection, or inducing an immune response against HBV.

The present disclosure also provides for the use of the aforementioned polypeptide, vector, vaccine, or composition to manufacture a medicament for use in treating HBV infection. The present disclosure also provides for the use of the aforementioned polypeptide, vector, vaccine, or composition to manufacture a medicament for use in protecting a subject from HBV infection. The present disclosure also provides for the use of the aforementioned polypeptide, vector, vaccine, or composition to manufacture a medicament for use in eliciting or inducing an immune response against HBV.

Provided herein is an HBV polypeptide comprising one or more episensus antigens, the episensus antigens having an amino acid sequence derived from HBV C, S, or P, the full-length sequence, a portion thereof, or any combination thereof.

Further provided herein is an immunogenic composition or vaccine comprising one or more pharma- ceutically acceptable carriers and one or more episensual antigens. Also provided herein is an immunogenic composition or vaccine comprising a vector capable of expressing one or more episensual antigens. In some embodiments, the HBV episensual antigen comprises two or more episensual sequences. In some embodiments, the HBV vaccine is a prophylactic vaccine. In some embodiments, the HBV vaccine is a therapeutic vaccine.

The present disclosure further provides a method of preventing or treating HBV infection in a subject, comprising administering to a subject in need thereof an effective amount of the immunogenic composition or vaccine described above. Further provided is a method of designing and producing an immunogenic composition or vaccine for a subject, comprising sequencing the HBV virus in the subject, selecting vaccine antigens designed to cover the diversity in the virus present in the subject, and inserting the vaccine antigens into a vector. Also provided herein is a method of treating HBV infection in a subject, comprising administering to a subject in need thereof an effective amount of the disclosed immunogenic composition or vaccine.

Also provided herein is a method of inducing an effector memory T cell response, the method comprising: (a) designing one or more episensous antigens; (b) producing an immunogenic composition or vaccine comprising a CMV backbone and a polynucleotide encoding the one or more episensous antigens; and (c) administering the vaccine to a subject in need thereof.

In certain embodiments, the episensus antigen of the methods provided herein comprises one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs:1-11 and SEQ ID NOs:14-36.
[The present invention 1001]
A polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1-11 or SEQ ID NO:14-36.
[The present invention 1002]
A polypeptide comprising two or more of the amino acid sequences set forth in SEQ ID NOs:16-36.
[The present invention 1003]
A polypeptide of the present invention comprising the amino acid sequence set forth in SEQ ID NO:19 or 29, the amino acid sequence set forth in SEQ ID NO:16, 21, 27, 28, or 34, and the amino acid sequence set forth in SEQ ID NO:20, 22, 23, 30, 31, 35, or 36.
[The present invention 1004]
The polypeptide of the present invention, comprising the amino acid sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4.
[The present invention 1005]
The polypeptide of the present invention, comprising the amino acid sequence set forth in SEQ ID NO:5 and the amino acid sequence set forth in SEQ ID NO:6.
[The present invention 1006]
The polypeptide of the present invention 1002, comprising the amino acid sequence set forth in SEQ ID NO:7 and the amino acid sequence set forth in SEQ ID NO:8.
[The present invention 1007]
The polypeptide of the present invention, comprising the amino acid sequence set forth in SEQ ID NO:9, the amino acid sequence set forth in SEQ ID NO:10, and the amino acid sequence set forth in SEQ ID NO:11.
[The present invention 1008]
The polypeptide of the present invention further comprises one or more of the amino acid sequences set forth in SEQ ID NOs:17 and 18.
[The present invention 1009]
A cytomegalovirus (CMV) vector comprising a polynucleotide comprising a sequence encoding one or more of the amino acid sequences set forth in SEQ ID NOs: 1-36.
[The present invention 1010]
The CMV vector of the present invention 1009, which is a human CMV (HCMV) vector or a rhesus CMV (RhCMV) vector.
[The present invention 1011]
The CMV vector of the present invention 1009 or 1010, which lacks the UL82 gene.
[The present invention 1012]
The CMV vector of any of claims 1009 to 1011, which lacks the UL128-UL130 gene region and also lacks the UL146-UL147 gene region.
[The present invention 1013]
The CMV vector of any one of claims 1009 to 1011, having an intact UL128-UL130 gene region and an intact UL146-UL147 gene region.
[The present invention 1014]
The CMV vector of any of claims 1009 to 1013, wherein the one or more amino acid sequences include one or more of SEQ ID NOs:1-11, SEQ ID NOs:14-15, and SEQ ID NOs:24-26.
[The present invention 1015]
The CMV vector of any of claims 1009 to 1013, wherein the one or more amino acid sequences comprise one or more of SEQ ID NOs: 16-23 and SEQ ID NOs: 27-36.
[The present invention 1016]
The CMV vector of any of claims 1009 to 1013, wherein the one or more amino acid sequences include two or more of SEQ ID NOs: 16 to 23 and SEQ ID NOs: 27 to 36.
[The present invention 1017]
The one or more amino acid sequences are
a) a sequence as set forth in SEQ ID NO: 19 or 29;
b) a sequence set forth in any one of SEQ ID NOs: 16, 21, 27, 28, or 34; and
c) a sequence set forth in any one of SEQ ID NOs: 20, 22, 23, 30, 31, 33, 35, or 36
The CMV vector of any one of claims 1009 to 1013, comprising:
[The present invention 1018]
The CMV vector of the present invention, wherein the one or more amino acid sequences further comprise one or more of SEQ ID NOs: 17 and 18.
[The present invention 1019]
The CMV vector of any of claims 1015 to 1018, wherein the sequence encoded by the polynucleotide is arranged to enhance expression.
[The present invention 1020]
The one or more amino acid sequences are
a) the sequence set forth in SEQ ID NO:3; and
b) the sequence set forth in SEQ ID NO:4
The CMV vector of any one of claims 1009 to 1013, comprising:
[The present invention 1021]
The one or more amino acid sequences are
a) the sequence set forth in SEQ ID NO:5; and
b) the sequence set forth in SEQ ID NO:6
The CMV vector of any one of claims 1009 to 1013, comprising:
[The present invention 1022]
The one or more amino acid sequences are
a) the sequence set forth in SEQ ID NO:7; and
b) the sequence set forth in SEQ ID NO:8
The CMV vector of any one of claims 1009 to 1013, comprising:
[The present invention 1023]
The one or more amino acid sequences are
a) the sequence set forth in SEQ ID NO:9;
b) the sequence set forth in SEQ ID NO:10; and
c) the sequence set forth in SEQ ID NO:11
The CMV vector of any one of claims 1009 to 1013, comprising:
[The present invention 1024]
A composition comprising two or more of the CMV vectors of the present invention according to any one of 1009 to 1023.
[The present invention 1025]
A Hepatitis B virus (HBV) vaccine comprising one or more of the polypeptides of the present invention 1001 to 1008.
[The present invention 1026]
A Hepatitis B virus (HBV) vaccine comprising a CMV vector and a polynucleotide encoding one or more HBV episensus antigens.
[The present invention 1027]
1026. The vaccine of claim 1026, wherein said CMV vector is a human CMV (HCMV) vector or a rhesus CMV (RhCMV) vector.
[The present invention 1028]
The vaccine of claim 1026 or 1027, wherein said CMV vector lacks the UL82 gene.
[The present invention 1029]
The vaccine of the present invention, wherein the polynucleotide encoding one or more HBV episensal antigens replaces the UL82 gene.
[The present invention 1030]
The vaccine of any of claims 1026 to 1029, wherein the CMV vector lacks the UL128 to UL130 gene region and the UL146 to UL147 gene region.
[The present invention 1031]
The vaccine of any of claims 1026 to 1029, wherein the CMV vector has an intact UL128 to UL130 gene region and an intact UL146 to UL147 gene region.
[The present invention 1032]
The vaccine of any of claims 1026 to 1031, wherein the polynucleotide encodes two or more HBV episensus antigens.
[The present invention 1033]
The vaccine of any of claims 1026 to 1031, wherein said one or more episensual antigens comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 36.
[The present invention 1034]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:1.
[The present invention 1035]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:2.
[The present invention 1036]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:3.
[The present invention 1037]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:4.
[The present invention 1038]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:5.
[The present invention 1039]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:6.
[The present invention 1040]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:7.
[The present invention 1041]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:8.
[The present invention 1042]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:9.
[The present invention 1043]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:10.
[The present invention 1044]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:11.
[The present invention 1045]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:14.
[The present invention 1046]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:15.
[The present invention 1047]
The vaccine of the present invention, wherein the episensus antigen comprises one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 16-23 and SEQ ID NOs: 27-36.
[The present invention 1048]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:24.
[The present invention 1049]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:25.
[The present invention 1050]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequence of SEQ ID NO:26.
[The present invention 1051]
The vaccine of any of claims 1026 to 1050, wherein the episensus antigen comprises one or more episensus sequences, the episensus sequences being derived from one or more of the C protein, the S protein, or the P protein.
[The present invention 1052]
1051. The vaccine of claim 1051, wherein said episensus antigen comprises an episensus sequence, said episensus sequence being derived from conserved regions of the C protein, the S protein, and the P protein.
[The present invention 1053]
13. The vaccine of claim 1051 or 1052, wherein said episensus sequences from said C protein, said S protein and said P protein are arranged to enhance expression.
[The present invention 1054]
The vaccine of any of claims 1051 to 1052, wherein the episensus antigen further comprises an episensus sequence, the episensus sequence being derived from one or more of the PreS1 domain, the PreS2 domain, and a non-conserved region of the P protein.
[The present invention 1055]
The vaccine of any of claims 1026 to 1054, wherein at least one of the protein sequences from which said episensus antigens are derived comprises one or more mutations.
[The present invention 1056]
The vaccine of the present invention, wherein the mutation is a deletion or substitution.
[The present invention 1057]
The vaccine of the present invention, wherein said mutation is a mutation of the P protein described in Table 1.
[The present invention 1058]
The vaccine of any of claims 1026 to 1057, wherein the episensus antigen comprises two or more episensus sequences.
[The present invention 1059]
The vaccine of the present invention, wherein the episensus antigen comprises two types of episensus sequences developed using HBV sequences originating from China.
[The present invention 1060]
1059. The vaccine of claim 1059, wherein said episensus antigen comprises the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4.
[The present invention 1061]
The vaccine of the present invention, wherein the episensus antigens include episensus sequences developed using HBV sequences from China and episensus sequences developed using HBV sequences from a global set.
[The present invention 1062]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6.
[The present invention 1063]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8.
[The present invention 1064]
The vaccine of the present invention, wherein the episensus antigen comprises three episensus sequences developed using HBV sequences from a worldwide set.
[The present invention 1065]
The vaccine of the present invention, wherein the episensus antigen comprises the amino acid sequences of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.
[The present invention 1066]
a) one or more HBV episensus antigens;
b) one or more polynucleotides encoding one or more HBV episensal antigens; or
c) one or more vectors comprising one or more polynucleotides encoding one or more HBV episensal antigens;
Including, HBV vaccine.
[The present invention 1067]
a) two or more HBV episensus antigens;
b) two or more polynucleotides encoding one or more HBV episensal antigens; or
c) two or more vectors containing one or more polynucleotides encoding one or more HBV episensal antigens;
1066. A vaccine according to the present invention comprising:
[The present invention 1068]
A composition comprising two or more of the HBV vaccines of the present invention Nos. 1026 to 1067.
[The present invention 1069]
The composition of the present invention 1068, wherein the first HBV vaccine encodes or comprises a first episensus antigen developed using an HBV sample originating from China, and the second HBV vaccine encodes or comprises a second episensus antigen developed using an HBV sample originating from China.
[The present invention 1070]
1069. The composition of claim 1069, wherein the first episensual antigen comprises the amino acid sequence of SEQ ID NO:3 and the second episensual antigen comprises the amino acid sequence of SEQ ID NO:4.
[The present invention 1071]
The composition of the present invention 1068, wherein the first HBV vaccine encodes or comprises a first episensus antigen developed using HBV sequences from China, and the second HBV vaccine encodes or comprises a second episensus antigen developed using HBV sequences from a worldwide set.
[The present invention 1072]
The composition of claim 1071, wherein the first episensual antigen comprises the amino acid sequence of SEQ ID NO:5 and the second episensual antigen comprises the amino acid sequence of SEQ ID NO:6.
[The present invention 1073]
The composition of claim 1071, wherein the first episensual antigen comprises the amino acid sequence of SEQ ID NO:7 and the second episensual antigen comprises the amino acid sequence of SEQ ID NO:8.
[The present invention 1074]
The composition of the present invention 1068, wherein the first HBV vaccine encodes or comprises a first episensus antigen developed using HBV sequences from a worldwide set, the second HBV vaccine encodes or comprises a second episensus antigen developed using HBV sequences from a worldwide set, and the third HBV vaccine encodes or comprises a third episensus antigen developed using HBV sequences from a worldwide set.
[The present invention 1075]
The composition of claim 1074, wherein the first episensual antigen comprises the amino acid sequence of SEQ ID NO:9, the second episensual antigen comprises the amino acid sequence of SEQ ID NO:10, and the third episensual antigen comprises the amino acid sequence of SEQ ID NO:11.
[The present invention 1076]
The vaccine of any one of claims 1026-1067 or the composition of any one of claims 1068-1075, wherein at least one episensus antigen is centred around an HBV sequence of Asian origin.
[The present invention 1077]
1076. The vaccine or composition of the present invention, wherein said at least one episensual antigen is centered on an HBV sequence originating from China.
[The present invention 1078]
The vaccine of any one of claims 1026-1067 or the composition of any one of claims 1068-1075, wherein at least one episensus antigen is centered on an HBV sequence from the United States.
[The present invention 1079]
The vaccine of any of claims 1026-1067 or the composition of any of claims 1068-1075, wherein at least one episensus antigen is centred around HBV sequences of European origin.
[The present invention 1080]
The vaccine of any of claims 1026-1067 or the composition of any of claims 1068-1075, wherein at least one episensus antigen is central to HBV sequences of US and European origin.
[The present invention 1081]
The vaccine of any of claims 1026-1067 or the composition of any of claims 1068-1075, wherein at least one episensus antigen is central to the worldwide set of HBV sequences.
[The present invention 1082]
The vaccine of any one of claims 1026 to 1067 or the composition of any one of claims 1068 to 1075, wherein at least one episensus antigen is central to the HBV genotype A epidemic.
[The present invention 1083]
The vaccine of any one of claims 1026 to 1067 or the composition of any one of claims 1068 to 1075, wherein at least one episensus antigen is central to the HBV genotype B epidemic.
[The present invention 1084]
The vaccine of any one of claims 1026 to 1067 or the composition of any one of claims 1068 to 1075, wherein at least one episensus antigen is central to the HBV genotype C epidemic.
[The present invention 1085]
The vaccine of any one of claims 1026-1067 or the composition of any one of claims 1068-1075, wherein at least one episensus antigen is central to the HBV genotype CB epidemic.
[The present invention 1086]
The vaccine of any one of claims 1026 to 1067 or the composition of any one of claims 1068 to 1075, wherein at least one episensus antigen is central to the HBV genotype D epidemic.
[The present invention 1087]
The vaccine of any one of claims 1026-1067 or the composition of any one of claims 1068-1075, wherein at least one episensus antigen is central to the HBV genotype DC epidemic.
[The present invention 1088]
The vaccine of any one of claims 1026-1067 or the composition of any one of claims 1068-1075, wherein at least one episensus antigen is central to the HBV genotype DE epidemic.
[The present invention 1089]
The vaccine of any one of claims 1026 to 1067 or the composition of any one of claims 1068 to 1075, wherein at least one episensus antigen is central to the HBV genotype E epidemic.
[The present invention 1090]
The vaccine of any one of claims 1026 to 1067 or the composition of any one of claims 1068 to 1075, wherein at least one episensus antigen is central to the HBV genotype F epidemic.
[The present invention 1091]
Any of the vaccines of claims 1026 to 1067 or any of the compositions of claims 1068 to 1075, wherein at least one episensus antigen is central to an HBV genotype other than genotypes A, B, C, CB, D, DC, DE, E, and F.
[The present invention 1092]
The vaccine of any one of 1026 to 1067 and 1076 to 1091 or the composition of any one of 1068 to 1075, wherein the vaccine is a prophylactic vaccine.
[The present invention 1093]
The vaccine of any of claims 1026 to 1067 and 1076 to 1091 or the composition of any of claims 1068 to 1075, wherein said vaccine is a therapeutic vaccine.
[The present invention 1094]
A vaccine according to any of claims 1026-1067 and 1076-1091 or a composition according to any of claims 1068-1075 further comprising a pharma- ceutically acceptable carrier.
[The present invention 1095]
A method for treating HBV in a subject, comprising administering to a subject in need thereof an effective amount of any of the polypeptides of the present inventions 1001-1008, any of the vectors of the present inventions 1009-1023, any of the vaccines of the present inventions 1025-1067 or 1076-1094, or any of the compositions of the present inventions 1024 or 1068-1075.
[The present invention 1096]
A method for protecting a subject from HBV infection, comprising administering to a subject in need thereof an effective amount of any of the polypeptides of the present inventions 1001-1008, any of the vectors of the present inventions 1009-1023, any of the vaccines of the present inventions 1025-1067 or 1076-1094, or any of the compositions of the present inventions 1024 or 1068-1075.
[The present invention 1097]
A method for causing or inducing an immune response against HBV in a subject, comprising administering to a subject in need thereof an effective amount of any of the polypeptides of the present inventions 1001-1008, any of the vectors of the present inventions 1009-1023, any of the vaccines of the present inventions 1025-1067 or 1076-1094, or any of the compositions of the present inventions 1024 or 1068-1075.
[The present invention 1098]
A polypeptide of any of claims 1001 to 1008, a vector of any of claims 1009 to 1023, a vaccine of any of claims 1025 to 1067 or 1076 to 1094, or a composition of any of claims 1024 or 1068 to 1075, for use in treating HBV.
[This invention 1099]
A polypeptide of any of claims 1001 to 1008, a vector of any of claims 1009 to 1023, a vaccine of any of claims 1025 to 1067 or 1076 to 1094, or a composition of any of claims 1024 or 1068 to 1075, for use in protecting a subject from HBV infection.
[The present invention 1100]
A polypeptide of any of claims 1001 to 1008, a vector of any of claims 1009 to 1023, a vaccine of any of claims 1025 to 1067 or 1076 to 1094, or a composition of any of claims 1024 or 1068 to 1075, for use in eliciting or inducing an immune response against HBV in a subject.
[The present invention 1101]
Use of any of the polypeptides of the inventions 1001-1008, any of the vectors of the inventions 1009-1023, any of the vaccines of the inventions 1025-1067 and 1076-1094, or any of the compositions of the inventions 1024 or 1068-1075 for the manufacture of a medicament for use in the treatment of HBV.
[The present invention 1102]
Use of any of the polypeptides of claims 1001-1008, any of the vectors of claims 1009-1023, any of the vaccines of claims 1025-1067 and 1076-1094, or any of the compositions of claims 1024 or 1068-1075 for the manufacture of a medicament for use in protecting a subject from HBV infection.
[The present invention 1103]
Use of any of the polypeptides of claims 1001 to 1008, any of the vectors of claims 1009 to 1023, any of the vaccines of claims 1025 to 1067 and 1076 to 1094, or any of the compositions of claims 1024 or 1068 to 1075 for the manufacture of a medicament for use in causing or inducing an immune response against HBV in a subject.
[The present invention 1104]
A method for designing and producing an HBV vaccine for subjects in a geographic region, comprising the steps of:
(a) designing one or more episensal antigens that effectively cover the diversity of HBV genotypes within the geographic region; and
(b) making one of the following:
an HBV vaccine comprising a CMV backbone and a nucleic acid encoding the episens antigen; or
An HBV vaccine comprising the episensus antigen.
[The present invention 1105]
1. A method of designing and producing an HBV vaccine for a subject, comprising:
(a) determining the amino acid sequence of the HBV virus in said subject;
(b) designing one or more episensal antigens to effectively cover the diversity of HBV genotypes present in the virus in the subject; and
(c) producing one of the following:
an HBV vaccine comprising a CMV backbone and a nucleic acid encoding the episens antigen; or
An HBV vaccine comprising the episensus antigen.
[The present invention 1106]
1. A method of inducing an effector memory T cell response, comprising the steps of:
(a) designing one or more episensal antigens;
(b) producing a vaccine comprising a CMV backbone and a nucleic acid encoding the one or more episens antigens; and
(c) administering said vaccine to a subject in need thereof.
[The present invention 1107]
A method of treating an HBV infection in a subject, comprising:
(a) producing an immunogenic composition comprising a CMV backbone and nucleic acid encoding one or more episensical antigens; and
(b) administering to the subject an effective amount of said immunogenic composition.
[The present invention 1108]
A method of preventing HBV infection in a subject, comprising:
(a) producing an immunogenic composition comprising a CMV backbone and nucleic acid encoding one or more episensical antigens; and
(b) administering to the subject an effective amount of said immunogenic composition.
[The present invention 1109]
2. A method of inducing an immune response against HBV in a subject, comprising:
(a) producing an immunogenic composition comprising a CMV backbone and nucleic acid encoding one or more episensical antigens; and
(b) administering to the subject an effective amount of said immunogenic composition.
[The present invention 1110]
1. A method of inducing an effector memory T cell response in a subject, comprising:
(a) producing an immunogenic composition comprising a CMV backbone and nucleic acid encoding one or more episensical antigens; and
(b) administering to the subject an effective amount of said immunogenic composition.
[The present invention 1111]
The method of any of claims 1104 to 1110, wherein said one or more episensous antigens comprise one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-11, SEQ ID NOs: 14-36.

Organization of the HBV genome. Unlike other small chronic viruses, such as HIV or HCV, which exhibit high mutation rates resulting in quasispecies, the small (3.2 kb) DNA genome of HBV is more restricted in its ability to mutate due to overlapping open reading frames (ORFs). Figures 2A-2B. Global distribution of 3041 HBV sequences available from the HBVdb. HBV genotypes are distributed worldwide. Only sequences for which the country sampled is known are shown on the map. 3A-3B. Distribution of HBV sequences available from the HBVdb in Asia and in China. In Asia, and especially in China, approximately 75% of the genotypes are genotypes B and C. HBV whole proteome coverage using one, two, and three episensous sequences. Figure 4A shows that one episensous sequence provides variable and limited coverage of the HBV genome (top panel). The coverage improves with the use of two episensous sequences (middle panel) or three episensous sequences (bottom panel). As shown by the number of perfect 9-mer matches, three episensus sequences provide better coverage than one or two episensus sequences. Figures 5A-5C. Whole proteome coverage using one, two, or three episensous sequences developed with the global HBV population. The use of two or three episensous sequences developed with the global HBV population substantially improves coverage for all major genotypes and provides comparable coverage for B and C compared to episensous sequences developed with the B and C HBV populations. Whole proteome coverage of HBV using two epicensus sequences for four conserved regions of the HBV genome. The hypervariable regions, encompassing the N-terminal 29 amino acids and the N-terminus of the P protein, are outlined. The hypervariable regions were excluded from further analysis. Coverage of potential T cell epitopes (PTEs) when one epicensus sequence is used in samples from China, in samples from outside China, and in a cohort of samples from around the world. The X-axis shows the percentage of coverage. The grey histogram shows the potential T cell epitope coverage by 1000 randomly selected natural strains. Coverage of potential T cell epitopes (PTEs) when two epicensus sequences are used in samples from China, samples from outside China, and a cohort of samples from around the world. The X-axis shows the percentage of coverage. The grey histogram shows the potential T cell epitope coverage by 1000 randomly selected natural strains. Comparison of coverage for different epicensus sequences. The 2_China-Worldwide_Epi (2_CHGL_epi) sequence provides a full-length sequence complete coverage of 0.809 (80.9%) for samples from around the world. The 2_CHGL_epi sequence provides a full-length sequence complete coverage of 0.879 (87.9%) for samples from China and a full-length sequence complete coverage of 0.772 (77.2%) for samples from outside China. Comparison of conserved region coverage in 1_D.vac, 1_C.ref, 1_CH_epi, 1_GL_epi, 2_CH_epi, 2_CHGL_epi, and 3_GL_epi in samples from China (Figure 9A, left), non-China (Figure 9A, middle), and a cohort of global samples (Figure 9A, right). Conserved region coverage in 1_D.vac, 1_GL_epi, and 2_CHGL_epi in all major genotypes is shown. Conserved region coverage for 1089 Chinese HBV sequences using 2_CHGL_epi sequences compared with wild-type genotype D sequences and 1_GL_epi sequences. Genotype D sequences are used in vaccines by Transgene and GlobeImmune. Conserved region coverage for all 3041 global HBV sequences. Genotype D sequences are used in vaccines by Transgene and GlobeImmune. 11A-11D. Figures 11A and 11B show episensous antigens for HBV genotype D developed for efficient epitope coverage in the United States and Europe; antigen constructs begin in Figure 11A and continue in Figure 11B. HBV genotype D episensous antigens are shown, derived from the core (C), PreS1 and PreS2, surface antigen (S), and polymerase (P) sequences in the order shown. Epicensous antigen variants have been developed with antigens derived from sequences with deletions in the transmembrane (TM) domain or with antigens that have been rearranged from the protein order in HBV to minimize aggregation and maximize expression. Figure 11C shows constructs for the same antigens, each containing a hemagglutinin (HA) epitope tag inserted at the C-terminus. FIG. 11D shows the expression of antigens by the viruses as measured by SDS polyacrylamide gel electrophoresis (SDS-page) followed by immunoblots illuminated with an antibody against the HA epitope tag incorporated into each construct. HBV genotype D episensor antigens derived from PreS1 and PreS2, surface antigen (S), polymerase (P), and/or core (C) sequences are shown. As shown, four of the antigens have deletions in the sequence derived from the P protein. A V5 epitope tag or a hemagglutinin (HA) epitope tag were incorporated into the construct; the V5 tag was inserted at the N'-terminus and the HA tag was inserted at the C'-terminus. Antigen expression by the viruses is shown as measured by SDS-page followed by immunoblot illuminated with antibodies against the V5 or HA epitope tags incorporated into each construct.

Detailed Description
I. Definitions
In the present specification and claims, various terms related to the aspects of this description are used. Such terms shall have their ordinary meaning in the art unless otherwise specified. Other terms that are specifically defined shall be interpreted in a manner consistent with the definitions set forth herein.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells, and the like. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives, and may be used synonymously with "and/or." As used herein, the terms "including" and "having" are used synonymously, and it is intended that the terms and variations thereof be construed as non-limiting.

As used herein, the term "about," when referring to measurable values such as amounts, durations, and the like, is intended to encompass variations of up to ±20% from the specified value, where such variations are appropriate for carrying out the disclosed methods. Unless otherwise specified, all numbers expressing quantities of ingredients, molecular weights, reaction conditions, and other properties used in the specification and claims are understood to be modified in all cases by the term "about." Accordingly, unless specified to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained. Without attempting to limit the application of the doctrine of equivalents to the scope of the claims, at a minimum, each numerical parameter should be construed in light of at least the number of recited significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters set forth in the broad scope of this disclosure are approximations, the numerical values set forth in the specific examples are described as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

The term "comprising" refers to the presence of the specified features, integers, steps, or components recited in the claim, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term "consisting essentially of" limits the scope of a claim to the specified materials or steps of the claimed subject matter and to materials or steps that do not materially affect the basic and novel characteristics of the claimed subject matter.

The word "substantially" does not exclude "entirely"; for example, a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted in the definitions provided herein, where appropriate.

As used above and in this disclosure, the term "effective amount" refers to an amount effective, at dosages and for periods of time necessary to achieve the desired result with respect to treating the disorder, disorder, or side effect in question. It is understood that the effective amount of a component will vary from patient to patient, not only depending on the particular vaccine, the component or composition selected, the route of administration, and the ability of the component to induce the desired result in the individual, but also depending on factors such as, for example, the condition or severity of the disorder being alleviated, the individual's hormone levels, age, sex, weight, condition of the patient, and the severity of the condition being treated, any concomitant medications or special diets planned for a particular patient thereafter, and other factors that one of skill in the art will recognize, and that appropriate dosages are at the discretion of the attending physician. Dosing regimens can be adjusted to improve therapeutic responses. An effective amount is also an amount in which any toxic or adverse effects of the component are outweighed by the therapeutically beneficial effects.

The term "administer" means either administering a compound or composition directly, or administering a prodrug, derivative, or analog that produces an equivalent amount of the active compound or substance in the body.

The terms "subject," "individual," and "patient" are used interchangeably herein and refer to an animal, e.g., a human, to which treatment, including prophylactic treatment, with the pharmaceutical compositions disclosed herein is provided. As used herein, the term "subject" refers to humans and non-human animals. The term "non-human animals" includes all vertebrates, including mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, horses, and non-mammals such as reptiles, amphibians, chickens, and turkeys.

The term "epitope" refers to an epitope-based consensus sequence, which is a sequence whose epitopes match as closely as possible with epitopes in a reference set with natural sequences. The terms "epitope" and "potential epitopes" refer to a k-letter sequence (typically k ranges from 8 to 12), often in the context of a k-letter subsequence of a very long natural or vaccine antigen sequence. T cells can recognize such peptides in an immune response.

The term "EpiGraph" refers to a computational strategy developed to generate sequences that provide optimal episensor sequences or to generate a set of sequences that, when combined, provide optimal coverage for a diverse population of viral sequences. The EpiGraph method has been previously described in PCT Application No. WO 2016/054654 A1, which are incorporated herein by reference in their entireties, and in Theiler, et al., Sci. Rep. 6:33987 (2016). The EpiGraph method generates a set of artificial, but intact, antigens with maximized coverage for potential T-cell epitopes (PTEs, typically 9-mer peptide sequences) found in a diverse population of viruses. EpiGraph is the next step beyond previous mosaic vaccine design methods. The graph theory-based EpiGraph method is computationally much more powerful than mosaic methods that use genetic algorithms, and therefore allows for improved PTE coverage by exploring substantially more combinations. Both the EpiGraph and mosaic methods generate protein antigens with greater coverage of T cell epitope diversity compared to natural strains. HBV mosaic, HIV mosaic, and HCV mosaic elicited broader and deeper cellular immune responses than combinations of natural strains or consensus immunogens. HBV mosaic and HIV mosaic are in Phase I clinical trials. In some embodiments, the EpiGraph algorithm is used to design "epicensus" sequences that correspond to conserved regions of HBV.

The term "epicensus sequence" refers to an amino acid sequence of an artificial antigen designed using the EpiGraph algorithm based on a population of HBV sequences. An epicensus sequence that is "central" for a population of HBV sequences is a computationally derived sequence that provides the greatest average epitope coverage for the population. An "epicensus antigen" is an antigen that comprises an epicensus amino acid sequence.

As used herein, the terms "treatment" or "therapy" (and variants thereof, including cure or mitigation) refer to the treatment of an infected human. As used herein, the term "treat" includes alleviating or reducing at least one of the deleterious or negative effects or symptoms of a disorder, disease, or disorder. The disorder, disease, or disorder may be an HBV infection.

As used herein, the terms "prevention" or "prophylaxis" refer to preventing a subject from becoming infected with, or reducing the risk of, a subject becoming infected with, or stopping the transmission of, an HBV or related virus, or reducing the risk of, a subject becoming infected with ...

"Pharmaceutically acceptable" refers to compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problematic complications, commensurate with a reasonable benefit/risk ratio.

As used herein, the terms "vaccine," "immunogenic compound," and "immunogenic composition" are used interchangeably to refer to a compound or composition that induces an immune response in a subject. A prophylactic vaccine provides some degree of protection against new infectious diseases. A therapeutic vaccine serves to treat an existing infectious disease.

II. Antigens
Provided herein are polypeptides comprising sequences derived from an HBV population. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NOs:1-11 or SEQ ID NOs:14-36. In some embodiments, the polypeptide comprises two or more of the amino acid sequences set forth in SEQ ID NOs:16-36. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NOs:19 or 29, an amino acid sequence set forth in SEQ ID NOs:16, 21, 27, 28, or 34, and an amino acid sequence set forth in SEQ ID NOs:20, 22, 23, 30, 31, 33, 35, or 36. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:5 and an amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:7 and an amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the polypeptide comprises an amino acid sequence set forth in SEQ ID NO:9, an amino acid sequence set forth in SEQ ID NO:10, and an amino acid sequence set forth in SEQ ID NO:11. In any of the foregoing embodiments, the polypeptide may further comprise one or more of the amino acid sequences set forth in SEQ ID NOs:17 and 18.

In some embodiments, the present disclosure provides immunogenic compositions or vaccines that include one or more of the aforementioned polypeptides. In some embodiments, the immunogenic compositions include one of the aforementioned polypeptides. In other embodiments, the immunogenic compositions include two or more of the aforementioned polypeptides, which may be provided as a single polypeptide or as two or more polypeptides.

In one embodiment, the polypeptide is an epicensus antigen, comprising a sequence derived from a wild-type HBV sequence using the EpiGraph method. In the EpiGraph algorithm, the natural sequence is characterized by a large graph of nodes, with each node corresponding to an epitope occurring in the natural sequence. A directed edge connects the two nodes if two corresponding epitope strings "match", meaning that the last k-1 characters in the first string match the first k-1 characters in the second string. When two strings match, they together form a string of length k+1. More generally, a path through this graph with nodes and edges corresponds to a single string that contains the k-mer substrings that correspond to each of the nodes in the graph. Each node is weighted according to how many sequences in the reference set exhibit the substring that corresponds to the node. The EpiGraph algorithm uses a dynamic programming scheme to find a path through this entire graph that maximizes the sum of these weights, and thus provides the greatest coverage.

In some embodiments, the episensual antigen is derived from the HBV C protein. In some embodiments, the episensual antigen is derived from the HBV Pol (P) protein or S protein, or another HBV protein. The episensual antigen may be derived from the full-length protein sequence, from a portion of the full-length protein sequence, or from any combination thereof. In some embodiments, the episensual antigen comprises epitopes from two or more of the HBV C, P, and S proteins. In some embodiments, the episensual antigen further comprises one or more epitopes from the PreS1 domain or the PreS2 domain. In some embodiments, the episensual antigen is derived from a conserved region of HBV. In some embodiments, the episensual antigen is derived from a conserved region of one or more of the HBV C, S, and P.

In some embodiments, the episensus antigen comprises sequences from two or more of the HBV C, P, and S proteins, which are rearranged from their order in the HBV genome. Rearrangement of sequences in the episensus antigen can result in more efficient or otherwise improved in vitro expression of the antigen.

In some embodiments, the episensual antigen is derived from an HBV protein having one or more mutations, such as, for example, an amino acid deletion or substitution. In some embodiments, the mutation inactivates at least one function present in the wild-type protein. For example, PreS1 can be inactivated by removing the myristoylation sequence at the N-terminus of the protein, or Pol can be inactivated by deleting or mutating the enzyme active site. In some embodiments, there is one or more mutations in the active site of HBV ribonuclease H (RNAse H) that result in loss of RNAse H activity. Exemplary mutations are shown in Table 1.

In some embodiments, episensus antigens are developed using a selected HBV population, resulting in episensus antigens that are central to the population. For example, the HBV population can be an HBV genotype present in a selected geographic location, such as Asia, North America, South America, Europe, Africa, or Australia. In some embodiments, the episensus antigen is central to a regional epidemic of HBV B in Asia, or more specifically in China. In other embodiments, the episensus antigen is central to a regional epidemic of HBV C in Asia, or more specifically in China. In some embodiments, the episensus antigen is central to a regional epidemic of HBV A in Asia, or more specifically in China. In some embodiments, the episensus antigen is central to a regional epidemic of HBV CB in Asia, or more specifically in China. In some embodiments, the episensus antigen is central to a regional epidemic of HBV D in Asia, or more specifically in China. In some embodiments, the episensus antigen is the focal point of a regional epidemic of HBV DC in Asia, or more specifically in China. In some embodiments, the episensus antigen is the focal point of a regional epidemic of HBV DE in Asia, or more specifically in China. In some embodiments, the episensus antigen is the focal point of a regional epidemic of HBV E in Asia, or more specifically in China. In some embodiments, the episensus antigen is the focal point of a regional epidemic of HBV F in Asia, or more specifically in China.

In other embodiments, the episens antigen is centered on a regional epidemic of multiple genotypes of HBV in North America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV A in North America. In some embodiments, the population episens antigen is centered on a regional epidemic of HBV B in North America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV C in North America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV CB in North America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV D in North America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV DC in North America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV DE in North America. In some embodiments, the epicensus antigen is a focal point of the regional epidemic of HBV E in North America. In some embodiments, the epicensus antigen is a focal point of the regional epidemic of HBV F in North America.

In other embodiments, the episens antigen is centered on a regional epidemic of multiple genotypes of HBV in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV A in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV B in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV C in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV CB in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV D in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV DC in South America. In some embodiments, the episens antigen is centered on a regional epidemic of HBV DE in South America. In some embodiments, the episensus antigen is a focal point of the regional epidemic of HBV E in South America. In some embodiments, the episensus antigen is a focal point of the regional epidemic of HBV F in South America.

In other embodiments, the episens antigen is centered on a regional epidemic of multiple genotypes of HBV in Europe. In some embodiments, the episens antigen is centered on a regional epidemic of HBV A in Europe. In some embodiments, the episens antigen is centered on a regional epidemic of HBV B in Europe. In some embodiments, the episens antigen is centered on a regional epidemic of HBV C in Europe. In some embodiments, the episens antigen is centered on a regional epidemic of HBV CB in Europe. In some embodiments, the episens antigen is centered on a regional epidemic of HBV D in Europe. In some embodiments, the population episens antigen is centered on a regional epidemic of HBV DC in Europe. In some embodiments, the episens antigen is centered on a regional epidemic of HBV DE in Europe. In some embodiments, the episens antigen is a focal point of the regional epidemic of HBV E in Europe. In some embodiments, the episens antigen is a focal point of the regional epidemic of HBV F in Europe.

In other embodiments, the episens antigen is central to the regional epidemic of multiple genotypes of HBV in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV A in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV B in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV C in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV CB in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV D in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV DC in Africa. In some embodiments, the episens antigen is central to the regional epidemic of HBV DE in Africa. In some embodiments, the epicensus antigen is a focal point of the regional epidemic of HBV E in Africa. In some embodiments, the epicensus antigen is a focal point of the regional epidemic of HBV F in Africa.

In other embodiments, the episens antigen is central to the regional epidemic of multiple genotypes of HBV in Australia. In some embodiments, the episens antigen is central to the regional epidemic of HBV A in Australia. In some embodiments, the episens antigen is central to the regional epidemic of HBV B in Australia. In some embodiments, the episens antigen is central to the regional epidemic of HBV C in Australia. In some embodiments, the episens antigen is central to the regional epidemic of HBV CB in Australia. In some embodiments, the episens antigen is central to the regional epidemic of HBV D. In some embodiments, the episens antigen is central to the regional epidemic of HBV DC. In some embodiments, the episens antigen is central to the regional epidemic of HBV DE. In some embodiments, the episens antigen is central to the regional epidemic of HBV E. In some embodiments, the epicensus antigen is the focus of a local epidemic of HBV F.

In other embodiments, the episensus antigens form the core of the HBV global set in the disclosed vaccines.

In some embodiments, the HBV episensus antigen is 1_CH_epi (SEQ ID NO:1), which was developed using 1044 HBV sequences from China. In some embodiments, the HBV episensus antigen is 1_GL_epi (SEQ ID NO:2), which was developed using a worldwide set of 3041 HBV sequences. In some embodiments, the episensus antigen is a combination of two or more individual episensus antigens. For example, in some embodiments, the episensus antigen includes 2_CH_epi, which includes two episensus antigens (SEQ ID NO:3 and SEQ ID NO:4) developed using 1044 HBV sequences from China. In some embodiments, the episensus antigens include 2_CHGL_epi, which includes a first episensus antigen, Epi1 (SEQ ID NO:5), developed using 1044 HBV sequences from China, and a second episensus antigen, Epi2 (SEQ ID NO:6), developed using a global set of 3041 HBV sequences, with Epi1 previously fixed to the solution. In other words, Epi2 is developed using the existing Epi1 and complements Epi1. In some embodiments, the episensus antigens include modified versions of 2_CHGL_epi, which includes Epi3 (SEQ ID NO:7) and Epi4 (SEQ ID NO:8). In some embodiments, the episensus antigens include 3_GL_epi, which includes three episensus antigens (SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11) developed using a global set of 3041 HBV sequences. In some embodiments, the episensus antigen comprises a native subtype D sequence (SEQ ID NO:12), GenBank Accession No. Y07587. In some embodiments, the episensus antigen comprises a native subtype C reference sequence (SEQ ID NO:13), GenBank Accession No. GQ358158. Each of the reference genes, Accession Nos. Y07587 and GQ358158, are incorporated herein by reference in their entirety. In some embodiments, the episensus antigen is a modified genotype D sequence, SEQ ID NO:14. In some embodiments, the episensus antigen is a permuted genotype D sequence, SEQ ID NO:15. In some embodiments, the episensus antigen comprises one or more of the sequences set forth in SEQ ID NOs:16-23 and SEQ ID NOs:27-36. In some embodiments, the HBV episensus antigen comprises two or more of the sequences set forth in SEQ ID NO:1 to SEQ ID NO:36.

Further provided herein is an immunogenic composition or vaccine comprising one or more of the aforementioned episensuous antigens. The episensuous antigen may be delivered as DNA, RNA, or as a polypeptide. In some embodiments, the vaccine comprises one antigen. In other embodiments, the vaccine comprises two or more antigens, which may be provided as a single polypeptide or as two or more polypeptides. In some embodiments, the vaccine comprises an episensuous antigen that provides efficient epitope coverage for a selected HBV genotype, such as a genotype present in a selected geographic location. The vaccines provided herein may be prophylactic or therapeutic vaccines. In some embodiments, the vaccine comprises one or more polypeptides and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the vaccine includes two episensus antigens developed with HBV sequences from China. In some embodiments, the vaccine includes episensus antigens comprising sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, which may be provided as a single polypeptide or as two polypeptides. In some embodiments, the vaccine includes episensus antigens developed with HBV sequences from China and episensus antigens developed with HBV sequences from the global set. In some embodiments, the vaccine includes episensus antigens comprising sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, which may be provided as a single polypeptide or as two polypeptides. In some embodiments, the vaccine includes episensus antigens comprising sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, which may be provided as a single polypeptide or as two polypeptides. In some embodiments, the vaccine includes three episensus antigens developed with HBV sequences from the global set. In some embodiments, the vaccine comprises an episensual antigen comprising the sequence set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, which may be provided as one polypeptide, two polypeptides, or three polypeptides.

III. Polynucleotides and Vectors
In some embodiments, the disclosure provides a polynucleotide sequence encoding a polypeptide. The polynucleotide may be DNA or RNA and may encode any of the aforementioned antigens. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in one or more of SEQ ID NOs:1-36. In some embodiments, the polynucleotide sequence is codon optimized for expression in a particular host.

In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1-11 or SEQ ID NO:14-36. In some embodiments, the polynucleotide encodes a polypeptide comprising two or more of the amino acid sequences set forth in SEQ ID NO:16-36. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:19 or 29, an amino acid sequence set forth in SEQ ID NO:16, 21, 27, 28, or 34, and an amino acid sequence set forth in SEQ ID NO:20, 22, 23, 30, 31, 33, 35, or 36. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:14. In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3 and the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5 and the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7 and the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the polynucleotide encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9, the amino acid sequence set forth in SEQ ID NO:10, and the amino acid sequence set forth in SEQ ID NO:11. In any of the foregoing embodiments, the polynucleotide may further encode a polypeptide comprising one or more of the amino acid sequences set forth in SEQ ID NOs:17 and 18.

In some embodiments, the present disclosure provides compositions, e.g., immunogenic compositions, that include two or more of the aforementioned polynucleotides.

In some embodiments, the present disclosure provides a vaccine comprising one or more of the aforementioned polynucleotides. In some embodiments, the vaccine further comprises a pharma- ceutically acceptable carrier or excipient.

Further provided herein is a vector comprising any of the aforementioned polynucleotides. Usable vectors include, but are not limited to, plasmids, bacterial vectors, and viral vectors. Viral vectors include cytomegalovirus vectors. An advantage of CMV vectors for use in therapeutic vaccine delivery is that they can be used to elicit specific CD8+ T cell responses and induce stronger and more durable responses. Vaccines based on these viral vectors have been shown in animal models to be able to eliminate viral infections (Hansen et al., Science 340:1237874 (2013)), and therefore these approaches are promising for therapeutic vaccines (also: Hansen et al, Science 351:714-720 (2016)).

Other viral vectors may include: poxviruses, including vaccinia Ankara virus and canarypox virus; adenoviruses, including adenovirus type 5 (Ad5); rubella virus; Sendai virus; rhabdovirus; alphavirus; and adeno-associated virus. Alternatively, vaccine antigens may be delivered as DNA, RNA, or proteins that are components of the vaccine. Epicensuous antigens may be compatible with essentially any mode of vaccine antigen delivery.

In some aspects, the present disclosure provides vector-based immunogenic compositions or vaccines in which an expression vector is used to deliver a nucleic acid encoding one or more of the antigens disclosed herein. For example, the expression vector can be a poxvirus, adenovirus, rubella virus, Sendai virus, rhabdovirus, alphavirus, or adeno-associated virus backbone.

In some embodiments, CMV vectors are used in the compositions and methods disclosed herein. In some aspects, chronic HBV infections are particularly suitable for immunotherapeutic intervention with CMV vectors.

First, the majority of individuals with mature immune systems rapidly mount effective immune responses and clear acute infections. Since this immune clearance is primarily mediated by cytokine-producing CD8+ T cells, it is clear that induction of active liver-resident effector CD8+ T cell (TEM) responses is crucial for HBV clearance. Thus, induction of persistent HBV-specific effector CD8+ T cells in the liver of CHB patients should be sufficient to control and ultimately clear HBV. The frequency of TEMs induced by CMV-based vectors is particularly high in the liver of inoculated rhesus macaques (RM) (Hansen et al., Nature 473:523-527 (2011), Supplementary Figure 1).

Second, unlike other small chronic viruses, such as HIV or HCV, which exhibit high mutation rates leading to quasispecies, the small (3.2 kb) DNA genome of HBV is more inhibited in its ability to mutate due to overlapping open reading frames (ORFs) (Figure 1).

Unfortunately, T cells induced by conventional vaccination approaches (e.g., peptide-based, protein-based, DNA, and even regimens involving both DNA and poxvirus vectors inducing heterologous prime-boost T cells) are no longer detectable in the periphery after several months, which may be too short to completely prevent the survival of residual HBV genomes that may have the potential to provide the source of viral reactivation. In contrast, only CMV vector-induced TEMs are persistently maintained in the circulation and liver for several years at high frequency (Hansen et al., Nature 2011).

In some embodiments, the vector comprises a human CMV (HCMV) vector or a rhesus CMV (RhCMV) vector, which comprises an HCMV or RhCMV backbone and one or more polynucleotides encoding antigens. The one or more polynucleotides may encode any of the aforementioned antigens. In some embodiments, the polynucleotide encodes two or more of the aforementioned antigens. In some embodiments, the vector comprises two or more polynucleotides, each polynucleotide encoding one or more of the aforementioned antigens.

The present disclosure further provides a composition comprising two or more vectors, each vector comprising one or more polynucleotides encoding one or more of the aforementioned antigens.

In some embodiments, the CMV vector lacks the UL82 gene, which encodes the tegument protein pp71. In some embodiments, the UL82 gene is replaced with one or more polynucleotides encoding one or more of the aforementioned antigens. In some embodiments, the CMV vector lacks the UL128-UL130 gene region. In some embodiments, the CMV vector lacks the UL146-UL147 gene region. In some embodiments, the CMV vector lacks the UL128-UL130 gene region and lacks the UL146-147 gene region. In some embodiments, the CMV vector has an intact UL128-UL130 gene region and an intact UL146-UL147 gene region. The UL128-UL130 gene region includes the UL128 gene, the UL130 gene, and any region between the UL128 gene and the UL130 gene. The UL146-UL147 gene region includes the UL146 gene, the UL147 gene, and any region between the UL146 gene and the UL147 gene.

In one aspect, the disclosure provides an immunogenic composition or vaccine comprising the aforementioned vector. The immune response elicited by the CMV vector is not affected by pre-existing anti-vector immunity, thus allowing the sequential use of the same vector for different antigens (Hansen et al., Nature Medicine 15:293-299 (2009)). The ability to superinfect is due, in part, to the virus's inhibitors of MHC-I-mediated antigen presentation to CD8+ T cells (Hansen et al., Science 328:102-106 (2010)). Since a large proportion of the population, including many CHB patients, are chronically infected with HCMV, superinfection is an important feature that allows the use of CMV vectors regardless of the recipient's CMV status. CMV vectors have unique immunological properties. A surprising and unexpected feature of some of the modified RhCMV/SIV vectors was the discovery that these vectors elicited CD8+ T cells that did not recognize any of the epitopes recognized by conventional MHC1a-restricted CD8+ T cells in response to SIV infection per se or in response to any other vector platform expressing SIV antigens. Nevertheless, as shown in PCT/US2016/017373 (see also Hansen et al., Science 2013, 2016), which is incorporated by reference in its entirety, CMV vector-elicited CD8+ T cells recognized 3-fold more peptides within a given antigen.

The underlying reason for this remarkable breadth was determined by analyzing the restriction elements for individual peptides of a given antigen (CMV, SIV, TB) in more than 100 animals, and this demonstrates a remarkable feature of the CMV vector: each of the peptides induced by the Rh 68-1 vector was presented by either MHC-II or MHC-E, where MHC-II is normally recognized by CD4+ T cells, and MHC-E is a non-polymorphic MHC-I molecule that normally binds the MHC-I-derived peptide VMAPRTLLL (VL9) and acts as a ligand for the NK cell inhibitory receptor NKG2A. (Note that all CD4+ T cells are conventional, i.e., MHC-II restricted.) Remarkably, the CD8+ T cell responses induced by the 68-1 strain of RhCMV to an overlapping pool of peptides covering the entire antigen could be completely blocked by the addition of VL9 (which inhibits MHC-E) or CLIP from the invariant chain (which inhibits MHC-II), thus proving that all CD8+ T cell epitopes are “unconventional” (Hansen et al., Science 2016). Although both MHC-II and MHC-E restricted CD8+ T cells have been observed occasionally in other infectious diseases and cancers, the presence of such T cells in large numbers in animals immunized with RhCMV is unprecedented and truly revolutionary. Importantly, RhCMV vector-induced MHC-II-restricted CD8+ T cells, MHC-E-restricted CD8+ T cells, and MHC-Ia-restricted noncanonical CD8+ T cells (see below) recognized SIV-infected CD4+ T cells, suggesting that nonconventional antigen presentation may occur in SIV-infected cells even if SIV is unable to prime such responses. Many chronic viruses, including HCV, upregulate HLA-E, presumably as a defense against NK cells, making this highly conserved MHC molecule a novel target for immunotherapy. (Although HLA-E expression in HBV-infected hepatocytes is less understood compared to HCV, high levels of circulating HLA-E have been found in chronic HBV carriers.) Thus, the ability to induce MHC-E-restricted CD8+ T cells opens the possibility of targeting HBV via this highly conserved restriction element.

Specific patterns of genetic modifications in CMV vectors are associated with different T cell responses. Non-conventional CD8+ T cells are induced by vectors lacking the UL128-UL130 and UL146-UL147 gene regions (Hansen et al., Science 2013, 2016, OHSU2346). In contrast, CD8+ T cell responses induced by natural RhCMV infection or against a UL128-UL130-repaired version of RhCMV (Rh68-1) are conventional, i.e., MHC-I restricted (Hansen et al., Science 2013). However, even conventional CD8+ T cell responses elicited by vectors with intact UL128-UL130 are still significantly broader than responses induced by non-CMV vectors (Hansen et al., Science 2013). Moreover, this broad conventional CD8+ T cell response is directed entirely to subdominant (non-canonical) epitopes, when the immune evasion gene US11 prevents the induction of T cells that recognize "canonical" (i.e., immunodominant) MHC-I epitopes (Hansen et al., Science 2013). Thus, genetically engineered CMV vectors can induce four distinct CD8+ T cell populations, each recognizing a non-overlapping set of epitopes and classified as follows: 1. unconventional, MHC-II restricted; 2. unconventional, MHC-E restricted; 3. conventional, MHC-I restricted, non-canonical (=subdominant); 4. conventional, MHC-I restricted, canonical (=immunodominant). The presence or absence of the UL128-UL130 and UL146-UL147 gene regions determines the switch of conventional to non-conventional CD8+ T cells, while canonical CD8+ T cells are induced in the absence of US11 by both vectors containing UL128-UL130 and UL146-UL147 and vectors lacking UL128-130 and UL146-UL147.

In some embodiments, the disclosure provides an immunogenic composition or vaccine comprising any of the aforementioned vectors and one or more polynucleotides encoding an antigen. The one or more polynucleotides may encode any of the aforementioned antigens. In some embodiments, the polynucleotide encodes two or more of the aforementioned antigens. The two or more polypeptides may be expressed by the same promoter or separate promoters. In some embodiments, the vector comprises two or more polynucleotides, each polynucleotide encoding one or more of the aforementioned antigens. In some embodiments, the vaccine comprises one or more of the aforementioned vectors and a pharmaceutically acceptable carrier or excipient.

III. Treatment Methods
In some embodiments, a method of treating HBV in a subject is provided, comprising administering to a subject in need thereof an effective amount of an immunogenic composition or vaccine comprising one or more of the aforementioned polypeptides, polynucleotides, vectors, or compositions. In some embodiments, the vaccine comprises one or more antigens or one or more polynucleotides encoding antigens, selected to effectively cover the diversity of HBV genotypes in a geographical area. In some embodiments, the vaccine is provided in the form of one or more polypeptides. In other embodiments, the vaccine is provided in the form of one or more polynucleotides, which may be provided as a recombinant viral vector. In any of the above embodiments, the vaccine may further comprise one or more pharma- ceutically acceptable carriers or excipients.

In some embodiments, methods of inducing or eliciting an immune response in a subject are provided. Such methods include administering to a subject in need thereof an effective amount of an immunogenic composition or vaccine comprising one or more of the aforementioned polypeptides, polynucleotides, vectors, or compositions. In some embodiments, the vaccine comprises one or more antigens or one or more polynucleotides encoding antigens selected to effectively cover the diversity of HBV genotypes within a geographic region. In some embodiments, the vaccine is provided in the form of one or more polypeptides. In other embodiments, the vaccine is provided in the form of one or more polynucleotides, which may be provided as a recombinant viral vector. In any of the aforementioned embodiments, the vaccine may further comprise one or more pharma- ceutically acceptable carriers or excipients.

Some embodiments include a method of treating HBV infection in a subject, comprising administering to a subject in need thereof an effective amount of an immunogenic composition or vaccine comprising one or more of the aforementioned polypeptides, polynucleotides, vectors, or compositions. In some embodiments, the vaccine comprises one or more antigens or one or more polynucleotides encoding antigens selected to effectively cover the diversity of HBV genotypes within a geographic region. In some embodiments, the vaccine is provided in the form of one or more polypeptides. In other embodiments, the vaccine is provided in the form of one or more polynucleotides, which may be provided as a recombinant viral vector. In any of the aforementioned embodiments, the vaccine may further comprise one or more pharma- ceutically acceptable carriers or excipients.

The present disclosure further provides a method of protecting a subject from HBV infection, comprising administering to a subject in need thereof an effective amount of an immunogenic composition or vaccine comprising one or more of the aforementioned polypeptides, polynucleotides, vectors, or compositions. In some embodiments, the vaccine comprises one or more antigens or one or more polynucleotides encoding antigens selected to effectively cover the diversity of HBV genotypes within a geographic region. In some embodiments, the vaccine is provided in the form of one or more polypeptides. In other embodiments, the vaccine is provided in the form of one or more polynucleotides, which may be provided as a recombinant viral vector. In any of the aforementioned embodiments, the vaccine may further comprise one or more pharma- ceutically acceptable carriers or excipients.

Some embodiments include a method of inducing an effector memory T cell response, the method including determining one or more episensous sequences, making a vaccine including a vector including one or more polynucleotides encoding the one or more episensous sequences, and administering the vaccine to a subject in need thereof. Some embodiments include a method of inducing an effector memory T cell response, the method including determining one or more episensous sequences, making a vaccine including one or more antigens having amino acid sequences of the one or more episensous sequences, and administering the vaccine to a subject in need thereof. In some embodiments, a method of inducing an effector memory T cell response is provided, wherein the one or more epi-sensus sequences include 1_CH_epi (SEQ ID NO:1), 1_GL_epi (SEQ ID NO:2), 2_CH_epi comprising the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, 2_CHGL_epi comprising the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, 2_CHGL_epi comprising the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, 3_GL_epi comprising the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, a native subtype D sequence (SEQ ID NO:12), a native subtype C reference sequence (SEQ ID NO:13), a modified genotype D sequence, which is SEQ ID NO:14, and a modified genotype D sequence, which is SEQ ID NO:15. A permuted genotype D sequence is NO:15. In some embodiments, a method of inducing an effector memory T cell response is provided, wherein the one or more episensous sequences include one or more of the sequences set forth in SEQ ID NOs:16-23 and SEQ ID NOs:27-36. In some embodiments, a method of inducing an effector memory T cell response is provided, wherein the one or more episensous sequences include two or more of SEQ ID NOs:1-36.

Some embodiments include a method of inducing an effector memory T cell response, the method comprising making an immunogenic composition or vaccine comprising one or more of the aforementioned polynucleotides encoding one or more episensual antigens, and administering the immunogenic composition or vaccine to a subject in need thereof. Some embodiments include a method of inducing an effector memory T cell response, the method comprising making an immunogenic composition or vaccine comprising one or more of the aforementioned episensual antigens, and administering the immunogenic composition or vaccine to a subject in need thereof. In some embodiments, a method of inducing an effector memory T cell response is provided, wherein the one or more epi-sensus sequences include 1_CH_epi (SEQ ID NO:1), 1_GL_epi (SEQ ID NO:2), 2_CH_epi comprising the amino acid sequences set forth in SEQ ID NO:3 and SEQ ID NO:4, 2_CHGL_epi comprising the amino acid sequences set forth in SEQ ID NO:5 and SEQ ID NO:6, 2_CHGL_epi comprising the amino acid sequences set forth in SEQ ID NO:7 and SEQ ID NO:8, 3_GL_epi comprising the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, a native subtype D sequence (SEQ ID NO:12), a native subtype C reference sequence (SEQ ID NO:13), a modified genotype D sequence, which is SEQ ID NO:14, and a modified genotype D sequence, which is SEQ ID NO:15. A permuted genotype D sequence is NO:15. In some embodiments, a method of inducing an effector memory T cell response is provided, wherein the one or more episensous sequences include one or more of the sequences set forth in SEQ ID NOs:16-23 and SEQ ID NOs:27-36. In some embodiments, a method of inducing an effector memory T cell response is provided, wherein the one or more episensous sequences include two or more of SEQ ID NOs:1-36.

In any of the above aspects, the immunogenic composition or vaccine can be a prophylactic vaccine or a therapeutic vaccine.

Recent developments in HBV vaccine research include the concept of a vaccine that induces effector memory T cells (TEMs) to prevent HBV infection. Unlike central memory T cells (TCMs) induced by conventional vaccine approaches, TEMs are persistently maintained in lymphoid tissues and extralymphoid effector sites and are ready to mediate antiviral effector functions, thus providing a persistent immune shield at the portal of viral entry and at the site of viral reactivation. The most suitable vector system to induce and maintain TEMs indefinitely is derived from CMV. Perhaps due to continuous, low-level reactivation and/or gene expression in persistently infected cells, CMV maintains a persistent, low-level immune stimulation just enough to maintain TEMs without causing T cell exhaustion.

In some embodiments, the immunogenic composition or vaccine may be used in a therapeutic vaccine design. For example, a vaccine may be designed using a k-means clustering strategy to identify a set of 6-10 antigen sequences that provide good epitope coverage in a human population infected with a highly variable pathogen, such as HBV. In some embodiments, the virus infecting the subject is sequenced and two or three vaccines (or one or more vaccines that include two or three polypeptides or include one or more polynucleotides encoding two or three polypeptides) that provide good epitope coverage of the infecting virus are delivered. Thus, efficient epitope coverage is provided while epitope mismatch between the vaccine and the infecting strain is minimized.

Some embodiments provided include immunogenic compositions or vaccines that include an HCMV backbone vector lacking some CMV gene region and a polynucleotide encoding an episensal antigen. In some embodiments, the HCMV backbone lacks the UL128-UL130 gene region (including the UL128 gene, the UL130 gene, and any region between the UL128 and UL130 genes) and lacks the UL146-UL147 gene region (including the UL146 gene, the UL147 gene, and any region between the UL146 and UL147 genes). Some embodiments may also include a deletion of the tegument protein pp71 (UL82) gene. (U.S. Patent Application Publication Nos. 2014/0141038; 2008/0199493; 2013/0142823; and International Publication WO/2014/138209).

In some embodiments, the disclosure provides any of the aforementioned polypeptides, polynucleotides, vectors, vaccines, or compositions for use in treating HBV infection. In some embodiments, the disclosure provides any of the aforementioned polypeptides, polynucleotides, vectors, vaccines, or compositions for use in protecting a subject from HBV infection.

The present disclosure further provides for the use of any of the aforementioned polypeptides, polynucleotides, vectors, vaccines, or compositions to manufacture a medicament for use in treating HBV infection. In some embodiments, the present disclosure further provides for the use of any of the aforementioned polypeptides, polynucleotides, vectors, immunogenic compositions, vaccines, or compositions to manufacture a medicament for use in protecting a subject from HBV infection.

Exemplary routes of administration of a polypeptide, polynucleotide, vector, vaccine, or composition described herein include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal routes. The term "parenteral" as used herein includes subcutaneous injection, intravenous, intramuscular, and intrasternal injection or infusion techniques. In certain embodiments, administration includes administration by a route selected from the following: oral, intravenous, parenteral, intragastric, intrathoracic, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumor, subcutaneous, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular.

Pharmaceutical compositions according to certain aspects of the invention are formulated so that the active ingredients contained therein are bioavailable when the composition is administered to a patient. The composition to be administered to a subject or patient may take the form of one or more dosage units, where, for example, a tablet may be a single dosage unit, and where the polypeptides, polynucleotides, vectors, vaccines, or compositions described herein are in liquid form, their containers may contain multiple dosage units. Actual methods for preparing such dosage forms are known or apparent to those of skill in the art; see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain an amount of the polypeptides, polynucleotides, vectors, vaccines, or compositions of the present disclosure effective to treat or prevent HBV in accordance with the teachings herein.

The composition may be in solid or liquid form. In some embodiments, the carrier is particulate, in which case the composition is, for example, in tablet or powder form. The carrier may be liquid, in which case the composition is, for example, an oral oil, an injectable liquid, or an aerosol, the latter being useful, for example, for inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where forms considered herein as either solid or liquid include semi-solid, semi-liquid, suspension, and gel forms.

The pharmaceutical composition may be formulated as a solid composition for oral administration into powder, granules, compressed tablets, pills, capsules, chewing gum, wafers, etc. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch, lactose, or dextrin, disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; flow agents, such as colloidal silicon dioxide;
A sweetening agent, for example, sucrose or saccharin; a flavoring agent, for example, peppermint, methyl salicylate, or orange flavoring; and a coloring agent. When the composition is in the form of a capsule, for example, a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier, such as polyethylene glycol or oil.

The composition may be in the form of a liquid, for example an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, to name two examples. If intended for oral administration, preferred compositions contain, in addition to the compounds of the present application, one or more of a sweetener, a preservative, a dye/colorant, and a flavor enhancer. Compositions intended to be administered by injection may contain one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.

Liquid pharmaceutical compositions, whether they are solutions, suspensions, or other forms, may contain one or more of the following adjuvants: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride solution, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol, or other solvents, which may act as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and tonicity adjusting agents, such as sodium chloride or dextrose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Saline is the preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile. Liquid compositions intended for either parenteral or oral administration should contain an amount of the polypeptide, polynucleotide, vector, vaccine, or composition disclosed herein such that an appropriate dosage will be obtained.

The composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment, or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. In compositions for topical administration, thickening agents may be present. If intended for transdermal administration, the composition may comprise a transdermal patch or an iontophoresis device. The pharmaceutical composition may be intended for rectal administration, for example, in the form of a suppository that dissolves in the rectum and releases the drug. Compositions for rectal administration may comprise an oleaginous base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

The composition may include various materials that modify the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell surrounding the active ingredient. The material that forms the coating shell is typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be placed in a gelatin capsule. The composition in solid or liquid form may include an agent that binds to the polypeptide, polynucleotide, vector, vaccine, or composition of the present disclosure and thereby aids in the delivery of the compound. Suitable agents that may perform in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes. The composition may consist essentially of a dosage unit that can be administered as an aerosol. The term aerosol is used to mean a variety of systems ranging from systems of colloidal nature to systems consisting of pressurized packages. Delivery may be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. To deliver the active ingredient, the aerosol can be delivered in a single-phase, two-phase, or three-phase system. The delivery of the aerosol includes the required containers, activators, valves, subcontainers, etc., which can be put together to form a kit. One of ordinary skill in the art can determine the preferred aerosol without undue experimentation.

It is understood that the compositions of the present disclosure also encompass carrier molecules (e.g., lipid nanoparticles, nanoscale delivery platforms, etc.) for the polynucleotides described herein.

The pharmaceutical compositions may be prepared by methods well known in the pharmaceutical arts. For example, compositions intended for administration by injection may be prepared by combining a composition comprising a polypeptide, polynucleotide, vector, vaccine, or composition described herein, and optionally one or more salts, buffers, and/or stabilizers, with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with molecules to promote dissolution or homogeneous suspension of the molecules in an aqueous delivery system.

In general, an appropriate dosage and treatment regimen is one that provides a sufficient amount of the composition to provide therapeutic and/or prophylactic benefit.

The composition is administered in an effective amount, which will vary depending on a variety of factors, including: the activity of the particular compound employed; the metabolic stability, and duration of action of the compound; the age, weight, general health, sex, and dietary habits of the subject; the mode and frequency of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and any treatments the subject is undergoing.

The following examples are provided to more fully describe the aspects described herein. The examples are intended to illustrate, but not to limit, the aspects.

All publications, patents, and patent applications cited herein are hereby incorporated by reference and may be employed in practicing the methods and compositions disclosed herein.

Example 1:
Identification of episensual antigens for use as therapeutic vaccines
Epicensus antigens for HBV were developed using the EpiGraph methodology, as described in PCT Application No. WO 2016/054654 A1, and Theiler, et al., Sci. Rep. 6:33987 (2016), which are incorporated by reference in their entireties. Tools for using the EpiGraph algorithm are also available at: hiv.lanl.gov/content/sequence/EPIGRAPH/epigraph.html.

While it is not practical to create a designer vaccine for each subject, it is feasible to sequence the virus from the subject and attempt to obtain a good match within a small reference set of vaccine options. A China-based HBV genotype B test population was used to identify vaccines that may provide therapeutic benefit. An Asia-based reference vaccine set, as well as an updated Asia-based genotype, and a global vaccine set were designed. Conserved regions of the HBV genome are found in the C protein (except for the first 29 amino acids at the N-terminus), S protein, and P protein. The highly variable region at the N-terminus of the P protein was excluded from consideration (see Figure 6).

Unlike a prophylactic vaccine, where it is unknown what virus a subject may be facing, with a therapeutic vaccine the sequence of the infecting virus is available and can be matched.

The selection of an antigen for use in an immunogenic composition or vaccine takes into account two factors: the antigen epitopes should match as many as possible with the epitopes of the virus infecting the subject, and there should be as little epitope mismatch as possible between the antigen and the virus infecting the subject, so that the vaccine response targets the appropriate epitopes as much as possible.

HBV major intragenotypic phylogenetic trees tend to have little clear structure. Rather, they are "radial," with very long external branches and very short, less clear internal branches near the base. Part of this structure appears to be due to intrasubtypic recombination. Although this is difficult to quantify, recombination certainly occurs relatively frequently and, by analogy with what has been observed for intersubtypic recombination, appears to be widespread. Given the structure of the tree, it would not be useful to use clustering on the tree alone to define a reference set for a potential vaccine, since intragenotypic relatedness is of limited significance in terms of epitopes. Instead, sequence relatedness should be examined by appropriate criteria, and reference sets should be selected based on the similarity of potential epitopes between natural strains and putative vaccine designs.

The Epigraph sequences were identified using a 9-mer of amino acids (see Figure 4A), but the method can be used with any length k-mer as a reference point, with k being the length of the putative potential epitope. In previous work with Mosaic, the optimal solution for the 9-mer was also near optimal for other nearby lengths (8, 10, 11, 12), so this is expected to be true for the EpiGraph method.

The epitope coverage of a) a vaccine containing one episensor sequence; b) a vaccine containing two episensor sequences; c) a vaccine containing three episensor sequences; d) a vaccine containing one native HBV genotype sequence; and e) a vaccine containing two native HBV genotype sequences is compared in Figures 4A-7B. In general, genotype-based vaccines provide superior coverage for the genotype, but poorer coverage for other genotypes. The data in Figures 5A-5C demonstrate that the bivalent and trivalent global EpiGraph sequences provide substantially improved coverage for all major genotypes, as well as comparable coverage for B and C compared to B- and C-specific vaccines. For example, as can be seen from Figures 5A-5C, coverage for genotype D is approximately 90% when three episensous antigens are used, in contrast to approximately 50% coverage with B-specific or C-specific vaccines, or even with B+C-specific vaccines.

Example 2:
Transient expression of episensal antigens
Antigens designed using the EpiGraph method to maximize epitope matching resemble natural sequences that no longer code for the original protein. Although the construction of these artificial amino acid sequences adheres to theoretical guidelines for expressing these sequences, the proteins may exhibit unexpected expression profiles or may not be able to express stable full-length proteins.

To assess the expression profile of these sequences in a mammalian cell environment, polynucleotides encoding the episensual antigens were synthesized and cloned for transient transfection using the methods described in WO 2016/054654, which is incorporated herein by reference in its entirety. Briefly, DNA encoding constructs were synthesized to contain cloning sites compatible with plasmid vectors (pcDNA3.1 and pOri) (Genscript, Piscataway, NJ). All inserts were codon-optimized for the host. Each construct was also modified to eliminate residual enzymatic activity of the original sequence, as described in Kulkarni et al. Vaccine (2011), which is incorporated herein by reference in its entirety. Plasmid vectors were linearized with compatible endonucleases and treated with calf intestinal phosphatase to prevent recircularization of the empty vector. The vector and insert fragments were separated by agarose gel electrophoresis to confirm the size of the digested fragments, and purified by PCR purification kit (Thermo Scientific) for ligation. The insert was ligated into the linearized vector at approximately 3:1 insert-to-vector ratio using a rapid ligation kit (Roche, Indianapolis, IN) for 15 min at room temperature, transformed into chemically competent E. coli (DH5-α), and plated on antibiotic selection plates. DNA from the resulting colonies was screened by digestion of the insert with restriction enzymes.

Clones containing the correct insert, each in the proper orientation relative to the vector's promoter and poly(A) sequences, were grown in liquid culture for purification of plasmid DNA. Actively growing, subconfluent 293T or HELA cells in 12-well tissue culture plates were added with 500 μl of fresh medium (DMEM 10% FBS) while liposomes were prepared. To generate liposomes containing plasmid DNA, 250 μl of serum-free medium was mixed with 500 ng of plasmid DNA, and 250 μl of serum-free medium was mixed with 2 ul of lipid (Lipofectamine 2000, Invitrogen). After 5 min of incubation at room temperature, these solutions were combined, mixed, and incubated for 20 min. DNA-containing liposomes formed during this process (500 μl) were added dropwise to the cultures and incubated for 12-16 h, after which the transfection mixture was replaced with fresh medium. After an additional day of incubation, cultures were harvested by scraping and centrifugation. The supernatant was removed by aspiration, and the cell pellet was lysed by resuspension in 100 μl of gel loading dye containing 5% SDS and 10% 2-mercaptoethanol and centrifugation through a QiaShred column (Qiagen, Valencia, CA).

Expression of episensus antigens was verified by SDS-polyacrylamide gel electrophoresis (SDS-page) and Western blotting using antibodies against the V5 epitope tag or hemagglutinin epitope tag incorporated in each construct. Briefly, NuPAGE 4-12% Bis-Tris gels were prepared and loaded with 20-50 ug total protein and electrophoresed at 110-120 volts for 90 minutes. Separated proteins were transferred to PVDF membranes by semi-dry or wet transfer at 30 volts for 90 minutes. Nonspecific binding was blocked for 60 minutes with 10% nonfat dry milk in Tris-buffered saline with 0.1% Tween-20 (PBS-T). HA (Sigma) or V5 (Invitrogen) antibodies were diluted in 5% nonfat dry milk solution and incubated with the membrane for 1 h, followed by 3 washes with TBS-T followed by 1 h incubation with horseradish peroxidase-conjugated goat anti-mouse (Invitrogen) secondary antibody at 1:10,000 dilution. Blots were then washed 3 times with TBS-T and developed using enzyme linked chemi-luminescence (ECL kit (Thermo-Pierce)) and visualized using a digital gel imaging system.

Example 3:
Incorporation of episensus antigens into CMV vector BAC constructs and expression from reconstructed viruses Epicensus antigens are designed to provide excellent coverage of the T cell epitopes that are representative of the viral sequence spectrum and HBV genotypes from which they are created.To use these antigens most effectively, the polynucleotides encoding the antigens are incorporated into CMV vectors, which have been shown to generate CD8 ⁺ T cell spectrums three times higher than competing platforms.The broad antigen presentation and lifelong expression profile of CMV vectors have demonstrated their ability to protect and cure SIV-infected rhesus macaques.The episensus antigen design algorithm combined with CMV vectors can provide even broader coverage of HBV within and between genotypes when widely applied to preventive vaccines.

Polynucleotides encoding episensal sequences, which have demonstrated expression in a transient transfection system, were introduced into the CMV backbone using BAC recombineering with galactokinase and kanamycin selection. (Warming et al. Nucleic Acids Research. 2005. 33(4):1-12; and Paredes and Yu Curr Protoc Microbiol. 2012. Feb; Chapter 14:Unit14E.4; both of which are incorporated by reference in their entirety.) Additionally, as shown in Figures 11A-11B, episensal sequences derived from HBV proteins or HBV protein domains, such as the core (C) protein, surface antigen (S) protein, PreS1 domain, PreS2 domain, and polymerase (P) protein, can be rearranged from the order in which they occur in wild-type HBV to improve expression.

BAC recombineering facilitates the manipulation of large DNA sequences using temperature- and metabolite-controlled recombination enzymes in the environment of SW105, an E. coli strain containing the parental BAC. Recombination is a two-step process consisting of step 1: insertion of a galactokinase (galK) sequence with the antibiotic resistance gene kanamycin (kan) into the target region, followed by step 2: replacement of the galK/kan cassette with the antigen of interest. Step 1 results in recombinants containing the galK/kan insert after positive selection with kan, and step 2 results in recombinants containing the antigen of interest after negative selection with 2-deoxy-galactose (DOG). For both steps, the sequence to be inserted is amplified by standard PCR from template DNA containing either the galK/kan cassette or the antigen of interest, using primers with long (50+ bp) homology arms to HCMV sequences flanking the insertion site.

To prepare bacterial cells for insertion of the galK/kan cassette (step 1), 5 mL cultures were grown overnight at 30°C in 2xYT or Terrific Broth containing 12.5 ug/mL chloramphenicol and diluted 1:50 the following morning. Bacteria were grown for approximately 2-4 more hours at 30°C (until OD ₆₀₀ = 0.5-0.6) and then heat shocked by shaking at 42°C for 15 minutes to induce recombinases. After induction, bacteria were pelleted (3000 rpm, 10 minutes, 4°C) and then washed three times with ice-cold water. E. coli cells were made electrocompetent to accept the PCR product and recombination competent to insert sequences into the target region of the BAC. The purified insert (300 ng) was combined with 40 μl of competent E. coli on ice, transferred to a 1 mm cuvette, and electroporated using a Bio-rad Gene Pulser Xcell. After electroporation, the bacteria were diluted by adding 5 mL of culture medium and allowed to recover by shaking at 30°C for 2 hours before plating onto chloramphenicol/kanamycin plates. The plates were incubated at 30°C for 2 days, and colonies were screened by restriction enzyme digestion and PCR for recombination events.

BAC constructs positive for the galK/kan insert were carried forward to the second step, in which the galK/kan cassette was replaced by a PCR fragment containing the antigen of interest by recombination. To prepare bacterial cells for replacing the galK/kan cassette in step 2, bacteria were grown and electroporated as described above. For recovery from electroporation, bacteria were diluted by adding 5 mL of culture medium and allowed to recover by shaking at 30°C for at least 4-4.5 hours. Cells were then pelleted (3000 rpm, 10 min, 4°C) and washed three times with 1x M9 medium before plating on M63 minimal medium plates supplemented with glycerol, leucine, biotin, DOG, and chloramphenicol. Colonies were then screened by PCR to confirm that the galK/kan cassette had been replaced with the antigen of interest. Positive clones were restreaked onto DOG plates, and BAC DNA was isolated for further characterization by restriction enzyme digestion and sequencing.

Virus reconstitution: To reconstitute the virus, BAC DNA was introduced into mammalian host cells permissive for virus growth. Briefly, BAC DNA was purified using an endotoxin-free plasmid DNA kit (Macherey-Nagel) and transfected into a flask of confluent primary human fibroblast cells (16-24 ug BAC DNA/T150) grown at 37°C and 5% _CO2 in DMEM containing 1x glutamax and 9% FBS. Transfection was performed using Lipofectamine 3000 according to the manufacturer's protocol (ThermoFisher). The next day, medium was changed and cells were monitored daily thereafter for plaque formation. After reaching maximum cytopathic effect (CPE), viral supernatants were harvested, clarified by centrifugation at 2500xg for 5 min at room temperature, and stored at -80°C. The remaining adherent cells were harvested with a cell scraper in DPBS, pelleted by centrifugation (2,500 rpm, 5 min), and stored at -80°C.

Expression of episensal antigens by viruses: Expression of episensal antigens was examined by SDS-polyacrylamide gel electrophoresis (SDS-page) followed by immunoblotting using either HBV-specific antibodies or antibodies against the V5 epitope tag or hemagglutinin (HA) epitope tag incorporated in each construct. Briefly, cell pellets were lysed by resuspension in radioimmunoprecipitation assay (RIPA) buffer, and proteins were quantified using a standard bicinchoninic acid assay (BCA). NuPAGE 4-12% Bis-Tris gels were prepared and loaded with 20-50 ug total protein and electrophoresed at 110-130 volts for 90 minutes. Separated proteins were transferred to PVDF membranes by semi-dry or wet transfer at 30 volts for 90 minutes or at 15 V overnight. Nonspecific binding was blocked for 60 min with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (PBS-T). Primary antibodies were diluted in 5% nonfat dry milk and incubated with the membrane for 1 h, followed by 3 washes with TBS-T followed by 1 h incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse secondary antibody. Blots were then washed 3 times with TBS-T and developed using an enzyme-linked chemiluminescence (ECL kit (Thermo-Pierce) and visualized using a digital gel imaging system.

Example 4:
Herd Epicensus Vaccine
The EpiGraph method was initially used to generate a set of vaccines containing polynucleotides encoding episensal antigens using CMV vectors, although other vaccine delivery systems can also be used.

A total of 58 strategies of different episensus antigens and antigen combinations were tested to determine the most comprehensive vaccine treatment. HBV samples from dbHBV were divided into three groups: samples from China, samples from outside China, and global samples. Global samples include a combination of samples from China and samples from outside China. A vaccine against native subtype D and a vaccine against native subtype C were used as controls. Vaccines with one, two, or three episensus antigens were tested for each group.

Controls included a vaccine containing a polynucleotide encoding the naturally occurring subtype D sequence (SEQ ID NO:12), GenBank Accession No. Y07587; and a vaccine containing a polynucleotide encoding the naturally occurring subtype C reference sequence (SEQ ID NO:13), GenBank Accession No. GQ358158. The vaccines containing polynucleotides encoding subtype D sequences and the vaccines containing polynucleotides encoding subtype C sequences were then compared to vaccines containing polynucleotides encoding: (a) 1_CH_epi (SEQ ID NO:1), which was developed using 1044 HBV sequences from China; (b) 1_GL_epi (SEQ ID NO:2), which was developed using a worldwide set of 3041 HBV sequences; (c) 2_CH_epi, which contains two episensus antigens (SEQ ID NO:3 and SEQ ID NO:4) developed using 1044 HBV sequences from China; (d) 2_CHGL_epi, which contains two episensus antigens - the first episensus antigen, Epi1 (SEQ ID NO:5), which was developed using 1044 HBV sequences from China. NO:5), and a second episensus antigen, Epi2 (SEQ ID NO:6), developed using a global set of 3041 HBV sequences with Epi1 previously fixed as the solution; (e) Epi7 and Epi8 (SEQ ID NO:7 and SEQ ID NO:8, respectively), which are variants of Epi1 and Epi2 that have been modified to induce better expression; and (f) 3_GL_epi, which includes three episensus antigens (SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11), developed using a global set of 3041 HBV sequences.

As shown in Figure 8, a vaccine with two episensual antigens, including polynucleotides encoding a first sequence developed using samples from China and a second sequence developed using the global set, provides approximately 88% coverage for Chinese sequences, approximately 77% coverage for non-Chinese sequences, and approximately 80% coverage for global sequences.

Example 5:
Epitope Coverage The HBV epitope coverage for vaccines containing several epicensus antigen sequences was analyzed computationally. The vaccine groups for the initial trials in CMV included: 1) one population epicensus antigen that is central to the Chinese genotype epidemic; 2) one population epicensus antigen that provides coverage for all HBV samples worldwide; 3) two population epicensus antigens that provide coverage for HBV samples from China; 4) two population epicensus antigens that provide coverage for all HBV samples worldwide; 5) three population epicensus antigens that provide coverage for all HBV samples worldwide; and 6) two population epicensus antigens: the first population epicensus antigen that provides coverage for HBV samples from China, and the second population epicensus antigen that provides coverage for HBV samples from the worldwide set.

Controls included a vaccine containing a polynucleotide encoding the naturally occurring subtype D sequence (SEQ ID NO:12), GenBank Accession No. Y07587; and a vaccine containing a polynucleotide encoding the naturally occurring subtype C reference sequence (SEQ ID NO:13), GenBank Accession No. GQ358158. The inventors then compared a vaccine comprising a polynucleotide encoding a subtype D sequence and a vaccine comprising a polynucleotide encoding a subtype C sequence with vaccines comprising polynucleotides encoding: (a) 1_CH_epi (SEQ ID NO:1), which was developed using 1044 HBV sequences from China; (b) 1_GL_epi (SEQ ID NO:2), which was developed using a worldwide set of 3041 HBV sequences; (c) 2_CH_epi, which contains two episensus antigens (SEQ ID NO:3 and SEQ ID NO:4) developed using 1044 HBV sequences from China; (d) 2_CHGL_epi, which contains two episensus antigens - the first episensus antigen, Epi1 (SEQ ID NO:5), developed using 1044 HBV sequences from China. NO:5), and a second episensus antigen, Epi2 (SEQ ID NO:6), developed using a global set of 3041 HBV sequences with Epi1 previously fixed as the solution; (e) Epi7 and Epi8 (SEQ ID NO:7 and SEQ ID NO:8, respectively), which are variants of Epi1 and Epi2 that have been modified to induce better expression; and (f) 3_GL_epi, which includes three episensus antigens (SEQ ID NO:9; SEQ ID NO:10; and SEQ ID NO:11), developed using a global set of 3041 HBV sequences.

As can be seen from Figures 9A-10B, the vaccine containing 2_CHGL_epi provides over 80 percent full coverage for sequences originating from China, over 75% full coverage for sequences originating from outside China, and 80% full coverage for sequences worldwide. These data demonstrate superior coverage for all HBV sequences compared to the control vaccine.

Example 6:
Second-generation HBV episensus antigens In many HBV genotypes, the N-terminal assembly domain (NTD) of HBV core (C) is involved in the assembly of the core particle, and the C-terminal domain is involved in packaging the pregenome/reverse transcriptase complex. The surface (S) protein is the product of a single open reading frame and is divided into three domains: PreS1, PreS2, and S. The polymerase (P) protein exhibits both DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase (reverse transcriptase) activity. The P protein replicates the HBV genome from a pregenome RNA template within the capsid. The P protein is composed of four domains: (1) the Terminal Protein (TP) domain, which is involved in the protein priming mechanism through a conserved tyrosine; (2) a non-conserved spacer domain; (3) a reverse transcriptase domain (RNA-dependent DNA polymerase (RT) and DNA-dependent DNA polymerase (active site: YMDD conserved motif)); and (4) an RNase H domain (ribonuclease H activity).

Epicensus antigens are designed to provide good coverage of T cell epitopes that are representative of the spectrum of viral sequences from which they are generated. HBV genotype D infection is one of the most prevalent HBV infections in the United States and Europe. A therapeutic vaccine containing an episensus (EpiD) antigen of genotype D or containing nucleotides encoding the EpiD antigen may be beneficial for patients who have been pre-screened for that particular genotype. Provided herein is an example of an episensus antigen of HBV genotype D (SEQ ID NO:14) that provides good coverage of HBV genotypes in the United States and Europe. In one variant, HBV genotype D episensous antigens derived from the core (C) protein, PreS1 and PreS2 domains, surface antigen (S) protein, and polymerase (P) protein were first used in the order found on the HBV genome: C-S-P ("CSP") (SEQ ID NO:14) and then rearranged to the order S-P-C ("SPC") (SEQ ID NO:15), as shown in Figures 11A and 11B. Additional antigen variants in which transmembrane domains 1-2 or 1-4 of the S protein are deleted to reduce aggregation and improve expression ("CSP ΔTM1-2", "CSP ΔTM1-4", "SPC ΔTM1-2", or "SPC ΔTM1-4") were generated by applying the EpiGraph method to the HBV protein sequence. Thus, HBV episensous antigens can also be derived from HBV protein sequences that include one or more of the aforementioned variants.

In addition, episensal antigens have been generated that are derived from P protein variants with mutations and/or deletions in the active site of the polymerase domain to reduce potential toxicity and improve safety. Examples of such episensal antigens are provided, for example, in SEQ ID NOs:7, 8, and 15. The mutations present in the sequences used to develop these episensal antigens are shown in Table 1 below. Other P protein variants with different mutations or deletions may also be used.

(Table 1) Mutations introduced into the HBV genotype D polymerase

The positions in the left column of the table refer to the number of amino acids in the full-length polymerase of HBV genotype D. The YMDD sequence is the active site of reverse transcriptase. (See Radziwill, et al., J Virol. 1990 Feb;64(2):613-20, which is incorporated by reference in its entirety.) The other four amino acids (D, E, D, D) are the active site of RNAse H. (See Tavis et al., PLoS Pathog. 2013 Jan;9(1):e1003125, which is incorporated by reference in its entirety.)

Expression of episensal antigens derived from these variant sequences was tested by SDS-page and Western blotting as described above (Figures 11A-11D). Removal of the transmembrane (TM) domain did not affect expression (Figure 11D).

No significant aggregation was observed with any of the EpiD antigen variants. Protein order in the antigen fusion was associated with efficient expression. Antigen fusion proteins permuted with SPC show significantly increased expression.

HBV episensal antigens may be derived from one or more of the following HBV proteins or HBV protein domains: core (C) protein, surface (S) protein, PreS1 protein, PreS2 protein, transmembrane domains 1-4 (TM1-4) of the S protein, determinants, and polymerase (P) protein. Epicensal antigens may be derived from various HBV proteins and HBV protein domains that have deletions or mutations and/or the episensal antigens may be rearranged from their order in HBV to improve expression or activity. By way of example, an episensor sequence derived from the core protein of HBV subtype D is provided in SEQ ID NO:16, an episensor sequence derived from the PreS1 protein of HBV subtype D is provided in SEQ ID NO:17, an episensor sequence derived from the PreS2 protein of HBV subtype D is provided in SEQ ID NO:18, an episensor sequence derived from the surface protein (S) of HBV subtype D is provided in SEQ ID NO:19, an episensor sequence derived from the polymerase (P) protein of HBV subtype D is provided in SEQ ID NO:20 or 22, and an episensor sequence derived from the polymerase (P) protein of HBV subtype D containing the mutations and deletions shown in Table 1 is provided in SEQ ID NO:23. An example of an epi-sensor sequence derived from the surface protein (S), polymerase protein (P), and core protein (C) of HBV subtype C, with antigen sequences in the order "SPC", is provided in SEQ ID NO:24. Examples of epi-sensor sequences derived from P proteins with larger deleted regions than those shown in Table 1 include: the epi-sensor sequence derived from the surface protein (S), polymerase protein (P) with a deletion of amino acids 612-838, and core protein (C) of HBV subtype C, provided in SEQ ID NO:25; and the epi-sensor sequence derived from the surface protein (S), polymerase protein (P) with a deletion of amino acids 601-827, and core protein (C) of HBV subtype D, provided in SEQ ID NO:26. Both SEQ ID NOs:25 and 26 are rearranged and provide antigen sequences in the order "SPC".

Further examples of epi-sensus sequences derived from P proteins with deleted regions are shown in Figure 12A and include deletions of amino acids 1-200, 201-400, or 401-600 of the P protein. Expression data for the constructs in Figure 12A are shown in Figure 12B.

Further examples of episensus sequences derived from the core protein of HBV are provided in SEQ ID NOs:27, 28, and 34. Further examples of episensus sequences derived from the surface protein of HBV are provided in SEQ ID NO:29. Further examples of episensus sequences derived from the polymerase protein of HBV are provided in SEQ ID NOs:30-32 and SEQ ID NOs:35-36.

The sequences of HBV proteins and HBV protein domains of any HBV subtype can be determined by sequence alignment with the sequences disclosed herein. For example, the complete genomic sequence of naturally occurring HBV subtype D is provided in GenBank Accession No. Y07587, which is incorporated herein by reference in its entirety. The P protein is encoded by nucleotide positions 1-1625 and 2309-3182 of the GenBank reference gene Y07587. The PreS protein is encoded by nucleotide positions 1-837 and 2850-3182 of the GenBank reference gene Y07587. The S domain is encoded by nucleotide positions 157-837 of the GenBank reference gene Y07587. The PreC/C protein is encoded by nucleotide positions 1816-2454 of the GenBank reference gene Y07587. The C protein is encoded by nucleotide positions 1903-2451 of the GenBank reference gene Y07587. In addition, the complete genomic sequence of native HBV subtype C is provided in GenBank Accession No. GQ358158, which is incorporated herein by reference in its entirety. The P protein is encoded by nucleotides 1-1623 and 2307-3215 of the GenBank reference gene GQ358158. The S protein is encoded by nucleotides 1-835 and 2848-3215 of the GenBank reference gene GQ358158. The PreC/C protein is encoded by nucleotides 1814-2452 of the GenBank reference gene GQ358158. The C protein is encoded by nucleotides 1901-2452 of the GenBank reference gene GQ358158.

Example 7:
Vaccine testing Vaccines containing computer-designed episensus antigens are tested in rhesus macaques (RM). T cell responses based on CMV are expected to be much broader than other vectors previously reported, and therefore cover a much higher percentage of sequences than other vectors previously reported. Therefore, even with a relatively small number of animals, there should be enough epitope responses to evaluate the impact of sequence diversity on the potential for cross-reactivity of responses. The number and strength of all responses to the vaccine are determined by using a peptide set matched to the vaccine. Once the target peptides are determined, the impact of natural diversity in each of the peptides that are responsive to the vaccine is determined using only those peptides that are positive in each animal. The natural variants tested are based on the diversity found in the reference panel. To evaluate the impact of epitope diversity on the reduction in the strength of recognition or the disappearance of recognition, non-parametric and computational resampling statistical methods are used as the main tool. However, these analyses are complemented by using generalized linear models, which are required to investigate the impact of more complex interactions on the cross-reactivity of T cell responses.

The vaccine group for initial testing in CMV includes vectors encoding: 1) one population epicensus antigen that is central to the Chinese genotype epidemic; 2) one population epicensus antigen that provides coverage for all global samples of HBV; 3) two population epicensus antigens that provide coverage for HBV samples from China; 4) two population epicensus antigens that provide coverage for all global samples of HBV; 5) three population epicensus antigens that provide coverage for all global samples of HBV; and 6) two population epicensus antigens: a first population epicensus antigen that provides coverage for HBV samples from China and a second population epicensus antigen that provides coverage for HBV samples from the global set; 7) an epicensus antigen (SEQ ID NO: 1) made with a truncated Pol sequence that provides coverage for HBV subtype C epitopes. NO:25), and 8) an episensus antigen made with a truncated Pol sequence that provides coverage for HBV subtype D epitopes (SEQ ID NO:26). Controls included a vaccine containing a polynucleotide encoding the native subtype D sequence (SEQ ID NO:12), GenBank Accession No. Y07587; and a vaccine containing a polynucleotide encoding the native subtype C reference sequence (SEQ ID NO:13), GenBank Accession No. GQ358158.

Up to ten cohorts of five rhesus macaques (RM) will be inoculated with 10 ⁶ PFU of HCMV vector as follows: up to eight cohorts will each receive one of vaccines 1-8 listed above; one cohort will receive a vaccine including a polynucleotide encoding native subtype D; and one cohort will receive a vaccine including a polynucleotide encoding native subtype C. Cohort 3 will receive a different tailored vaccine vector in addition to Epicensus, and cohort 4 will receive both tailored vaccine vectors in addition to Epicensus.

Rhesus macaques (RM) are inoculated subcutaneously at days 0 and week 12 and followed longitudinally for 1 year. Because vaccination with HCMV vectors is not affected by pre-existing anti-RhCMV immunity, animals naturally infected with RhCMV are used in these experiments. Intracellular cytokine analysis (ICS) by flow cytometry is used to determine CD4+ and CD8+ T cell responses (which includes all vaccine-induced responses) to individual consecutive 15-mer peptides containing the vaccine sequence within the vaccine insert administered to each animal. It is then determined whether these epitope-specific T cells recognize epitope variants in both targeted and non-targeted strains. For peptides that show responses against strain-specific epitopes, the strength, functional avidity, and functional characteristics (IFN-γ, TNF-α, IL-2, and MIP-1β production, and CD107 externalization) of the responses against the "parental" (vaccine insert sequence) peptide variants are compared to determine the degree of functional cross-reactivity. In selected examples, truncation analysis is used to identify core epitopes for similarity comparison analysis. To determine the percentage of MHC-II-restricted CD8+ T cells present, MHC-I-specific and MHC-II-specific "blocking" mAbs, as well as CLIP, an invariant chain-derived, MHC-II-specific binding peptide, are used to inhibit influenza-specific CD8+ T cell responses in PBMCs.

Although specific embodiments have been illustrated and described, it will be readily understood that various embodiments described above may be combined to provide further embodiments, and in so doing, various modifications may be made without departing from the spirit and scope of the present invention.

All U.S. patents, U.S. patent application publications, U.S. patent applications, non-U.S. patents, non-U.S. patent applications, and non-patent literature referenced herein and/or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 62/893,546, filed August 29, 2019, and U.S. Provisional Patent Application No. 62/941,125, filed November 27, 2019, are incorporated by reference in their entirety unless expressly specified otherwise. Aspects of the present embodiments may be modified to employ concepts from various patents, applications, and publications as necessary to provide further embodiments.

These and other modifications can be made to the present embodiments in light of the above detailed description. Generally, in the appended claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims (8)

A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. A cytomegalovirus (CMV) vector comprising a polynucleotide comprising a sequence encoding the amino acid sequence set forth in SEQ ID NO:15. 3. The CMV vector of claim 2 , which is a human CMV (HCMV) vector or a rhesus CMV (RhCMV) vector. 4. The CMV vector of claim 2 or 3 , which lacks the UL82 gene. The CMV vector according to any one of claims 2 to 4 , which lacks the UL128 to UL130 gene region and the UL146 to UL147 gene region. The CMV vector according to any one of claims 2 to 4 , having an intact UL128-UL130 gene region and an intact UL146-UL147 gene region. A composition comprising two or more CMV vectors according to any one of claims 2 to 6 . A polynucleotide encoding the amino acid sequence set forth in SEQ ID NO:15.

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